Composition for introducing nucleic acid complexes into higher eucaryotic cells

ABSTRACT

A composition for the transfection of higher eucaryotic cells, comprising complexes of nucleic acid, a substance having an affinity for nucleic acid and optionally an internalizing factor, contains an endosomolytic agent, e.g. a virus or virus component, which may be conjugated. The endosomolytic agent, which is optionally part of the nucleic acid complex, is internalized into the cells together with the complex and releases the contents of the endosomes into the cytoplasm, thereby increasing the gene transfer capacity. Pharmaceutical preparations, transfection kits and methods for introducing nucleic acid into higher eucaryotic cells by treating the cells with the composition are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.08/449,754, filed May 25, 1995 now U.S. Pat. No. 6,077,663, which is acontinuation-in-part of U.S. application Ser. No. 07/937,788, filed Sep.2, 1992, now abandoned which is a continuation-in-part of U.S.application Ser. No. 07/864,759, filed Apr. 7, 1992, which is acontinuation-in-part of U.S. application Ser. No. 07/827,102, filed Jan.30, 1992, now abandoned which is a continuation-in-part of U.S.application Ser. No. 07/767,788, filed Sep. 30, 1991 now abandoned. Thepresent application is also a continuation-in-part of U.S. applicationSer. No. 07/827,103, filed Jan. 30, 1992, now abandoned and is acontinuation-in-part of U.S. application Ser. No. 07/768,039, filed Sep.30, 1991 now abandoned. The contents of each of these relatedapplications is fully incorporated by reference herein.

FIELD OF THE INVENTION

The invention is in the field of DNA technology. In particular, theinvention relates to new compositions which can be used for theintroduction of nucleic acids into higher eucaryotic cells.

BACKGROUND OF THE INVENTION

There is a need for an efficient system for introducing nucleic acidinto living cells particularly in gene therapy. Genes are transferredinto cells in order to achieve in vivo synthesis of therapeuticallyeffective genetic products, e.g. in order to replace the defective genein the case of a genetic defect. “Conventional” gene therapy is based onthe principle of achieving a lasting cure by a single treatment.However, there is also a need for methods of treatment in which thetherapeutically effective DNA (or mRNA) is administered like a drug(“gene therapeutic agent”) once or repeatedly as necessary. Examples ofgenetically caused diseases in which gene therapy represents a promisingapproach are hemophilia, beta-thalassaemia and “Severe Combined ImmuneDeficiency” (SCID), a syndrome caused by the genetically induced absenceof the enzyme adenosine deaminase. Other possible applications are inimmune regulation, in which humoral or intracellular immunity isachieved by the administration of functional nucleic acid which codesfor a secreted protein antigen or for a non-secreted protein antigen,which may be regarded as a vaccination. Other examples of geneticdefects in which a nucleic acid which codes for the defective gene canbe administered, e.g. in a form individually tailored to the particularrequirement, include muscular dystrophy (dystrophin gene), cysticfibrosis (cystic fibrosis transmembrane conductance regulator gene),hypercholesterolemia (LDL receptor gene). Gene therapy methods are alsopotentially of use when hormones, growth factors or proteins with acytotoxic or immune-modulating activity are to be synthesized in thebody.

Gene therapy also appears promising for the treatment of cancer byadministering so-called “cancer vaccines”. In order to increase theimmunogenicity of tumor cells, they are altered to render them eithermore antigenic or to make them produce certain immune modulatingsubstances, e.g. cytokines, in order to trigger an immune response. Thisis accomplished by transfecting the cells with DNA coding for acytokine, e.g. IL-2, IL-4, IFN gamma, TNF alpha. To date, most genetransfer into autologous tumor cells has been accomplished viaretroviral vectors.

The technologies which are hitherto most advanced for the administrationof nucleic acids in gene therapy, make use of retroviral systems fortransferring genes into the cells (Wilson et al., 1990, Kasid et al.,1990). However, the use of retroviruses is problematic because itbrings, at least to a small degree, the danger of side effects such asinfection with the virus (by recombination with endogenous viruses orcontamination with helper viruses and possible subsequent mutation intothe pathogenic form) or the formation of cancer. Moreover, the stabletransformation of the somatic cells in the patient, as achieved withretroviruses, is not desirable in every case because it might make thetreatment difficult to reverse, e.g. if side effects occur. Moreover,with this type of therapy, it is difficult to obtain a high enough titerto infect enough cells.

Nucleic acids as therapeutically effective substances are also used toinhibit specific cell functions, e.g. antisense RNAs and DNAs haveproved effective in the selective inhibition of specific gene sequences.Their mode of activity enables them to be used as therapeutic agents forblocking the expression of certain genes (such as deregulated oncogenesor viral genes) in vivo. It has already been shown that short antisenseoligonucleotides can be imported into cells and exert their inhibitingeffect therein (Zamecnik et al., 1986), even if their intracellularconcentration is low, caused, inter alia, by their restricted uptake bythe cell membrane as a result of the strong negative charge of thenucleic acids. Another approach to the selective inhibition of genes isthe use of ribozymes. Again there is the need to ensure the highestpossible concentration of active ribozymes in the cell, transportationinto the cell being one of the limiting factors.

Application of gene therapy for achieving intracellular immunityinvolves transduction of genes which protect against viruses, so-called“protective genes”, e.g. transdominant mutants of genes coding for viralproteins, or DNA molecules coding for so-called RNA decoys.

There is consequently a need for methods of enabling the transfer andexpression of DNA into the cell.

Various techniques are known for gene transformation of mammalian cellsin vitro, but their use in vivo is limited (these include theintroduction of DNA by means of liposomes, electroporation,microinjection, cell fusion, DEAE-dextran or the calcium phosphateprecipitation method).

In recent times, recombinant viral vectors have been developed to bringabout the transfer of genes by using the efficient entry mechanisms oftheir parent viruses, this strategy was used in the construction ofrecombinant retroviral and adenoviral vectors in order to achieve ahighly efficient gene transfer in vitro and in vivo (Berkner, 1988).Despite their efficiency, these vectors are subject to restrictions interms of the size and construction of the DNA which is transferred.Furthermore, these agents represent safety risks in view of the transferof the viable viral gene elements of the original virus.

In order to circumvent these restrictions, alternative strategies forgene transfer have been developed, based on mechanisms which the celluses for the transfer of macromolecules. One example of this is thetransfer of genes into the cell via the extremely efficient route ofreceptor-mediated endocytosis (Wu and Wu, 1987, Wagner et al., 1990 andEP-A1 0388 758, the disclosure of which is hereby referred to). Thisapproach uses bifunctional molecular conjugates which have a DNA bindingdomain and a domain with specificity for a cell surface receptor (Wu andWu, 1987, Wagner et al., 1990). If the recognition domain is recognizedby the cell surface receptor, the conjugate is internalized by the routeof receptor-mediated endocytosis, in which the DNA bound to theconjugate is also transferred. Using this method, it was possible toachieve gene transfer rates at least as good as those achieved with theconventional methods (Zenke et al., 1990). Furthermore, it has beenshown that the activity of a nucleic acid, e.g. the inhibitory effect ofa ribozyme, is not impaired by the transport system.

The PCT application WO 91/17773 relates to a system for transportingnucleic acids with a specific activity for T-cells. This system makesuse of cell surface proteins of the T-cell lineage, e.g. CD4, thereceptor used by the HIV virus. The nucleic acid to be imported iscomplexed with a protein-polycation conjugate, the protein component ofwhich, i.e. the recognition domain, is a protein capable of binding tothe T-cell surface protein, e.g. CD4, and cells which express thissurface protein are brought into contact with the resultingprotein-polycation/nucleic acid complexes. It has been shown that DNAtransported into the cell by means of this system is expressed in thecell.

One feature common to both inventions is that they use specific cellfunctions to enable or facilitate the transfer of nucleic acid into thecell. In both cases, the uptake mechanisms take place with theparticipation of recognition domains which are termed “internalizingfactors” within the scope of the present invention. This term denotesligands which, being cell-type-specific in the narrower or wider sense,bind to the cell surface and are internalized, possibly with thecooperation of other factors (e.g. cell surface proteins). (In the caseof the two inventions mentioned above, the internalizing factor istransferrin or a protein which binds to a T-cell surface antigen, e.g.an anti-CD4 antibody). The internalizing factor is conjugated with asubstance of a polycationic nature which, by virtue of its affinity withnucleic acids, forms a strong association between the internalizingfactor and the nucleic acid. (Substances of this kind are hereinafterreferred to as “substances with an affinity for nucleic acid” or withregard to DNA, “DNA binding domain”. If a substance of this kind forms abond between the nucleic acid and an internalizing factor it ishereinafter referred to as a “binding factor”).

In the course of these two inventions the optimum uptake of nucleic acidinto the cell was achieved when the ratio of conjugate to nucleic acidwas such that the internalizing factor-polycation/nucleic acid complexeswere approximately electroneutral. Starting from this observation, themethods which use internalizing factor-binding factor/nucleic acidcomplexes to introduce nucleic acids into higher eucaryotic cells wasimproved.

A method for improving the efficiency of systems in which the uptake ofnucleic acids is carried out by means of internalizing factors, wasdescribed by Wagner et al., 1991a. In this method, the quantity ofnucleic acid taken up into the cell is not reduced if part of thetransferrin-polycation conjugate is replaced by non-covalently bound(“free”) polycation; in certain cases, this may even increase the DNAuptake considerably. Investigations into the molecular state oftransferrin-polycation-plasmid DNA complexes produced with optimumratios of DNA/conjugate showed that the plasmid DNA in the presence ofthe conjugates is condensed into toroidal structures (resemblingdoughnuts) with a diameter of about 80 to 100 nm).

Experiments conducted with proteins binding to T-cells as internalizingfactor produced similar results.

The addition of “free” substances with an affinity for nucleic acid alsoresults in an increase in the efficiency of the introduction system evenwhen other binding factors are used.

The complexes described by Wagner et al., 1991a, which are taken up intohigher eucaryotic cells via endocytosis by means of internalizingfactor, contain nucleic acid complexed with a conjugate of internalizingfactor and binding factor. In addition, the complexes contain one ormore substances with an affinity for nucleic acid which may possibly beidentical to the binding factor, in a non-covalently bonded form, suchthat the internalization and/or expression of the nucleic acid achievedby means of the conjugate is increased, which would appear to be dueprimarily to a condensing effect but might possibly be due to othermechanisms.

Even if the rates of expression of the imported nucleic acid could beincreased by this method, it is still subject to restrictions. Thepracticality of this system in a given context is not solely determinedby the presence of the cell surface receptor relevant to the system; thelimitations associated with the use of this system are presumably aresult of the fact that the conjugate-DNA complexes internalized inendosomes enter the lysosomes, where they are enzymatically degraded. Inorder to increase the proportion of nucleic acid which reaches the cellnucleus and is expressed there, as intended, attempts were made, inexperiments preceding this invention, to carry out the transfection ofthe cells in the presence of substances which inhibit the enzymaticactivity in the lysosomes, so-called lysosomatropic substances. By usingthis strategy, augmented expression of transferred DNA was achieved;however, the reactions achieved were highly variable, depending on thesubstance used; selected lysosomatropic substances brought about anincrease in gene transfer, whereas others actually inhibited it. Thus,for example, it was found that the effective transfer of DNA depends onthe presence of the weak base chloroquine (Zenke et al., 1990, Cotten etal., 1990). This effect achieved by means of chloroquine may not, or notexclusively, be due to the fact that chloroquine increases the pH in thelysosomes; it was found, from a number of different experiments, thatother substances which, like chloroquine, have the ability to modulatepH, such as monensin, ammonium chloride or methylamine, could notreplace chloroquine and in some experiments some of these substanceseven showed an inhibiting effect. It was further found that varioustarget cells show different responses to the same substance having alysosomatropic activity.

Since gene transfer by the physiological route, as represented byreceptor-mediated endocytosis using nucleic acid complexes, has majoradvantages (non-toxic mechanism of passage through the cell membrane;the possibility of administering biologically active nucleic acids, suchas nucleic acids which specifically inhibit genes, or cellularfunctions, on a repeated or continuous basis; the possibility ofcell-specific targeting; the possibility of producing the conjugates inlarge quantities), there is a need to make this system more efficient.

SUMMARY OF THE INVENTION

The present invention is directed to a composition for the transfectionof higher eucaryotic cells with a complex of nucleic acid and asubstance having affinity for nucleic acid, which substance isoptionally coupled with an internalizing factor for the cells,characterized in that the composition contains an agent which has theability of being internalized into the cells, either per se or as acomponent of the nucleic acid complex, and of releasing the contents ofthe endosome, in which the complex is located after entering the cell,into the cytoplasm.

The present invention also relates to a nucleic acid complex useful asconstituent of the composition of the invention, wherein the complexcomprises one or more nucleic acids to be expressed in the cell, anendosomolytic agent which originally has a nucleic acid binding domainor which is bound to a substance having affinity for nucleic acid, andwherein the complex optionally further comprises an internalizing factorwhich is bound to a substance having affinity for nucleic acid.

The invention also relates to an endosomolytic peptide useful as aconstituent of the composition of the invention, characterized in thatit has an endosomolytic domain and a nucleic acid binding domain.

The invention also relates to a process of preparing a conjugate of theinvention which is useful for enhancing the uptake of nucleic acid intohigher eucaryotic cells, characterized in that a virus or a(poly)peptidic endosomolytic agent and a polyamine are enzymaticallycoupled in the presence of a transglutaminase.

The invention also relates to a process of preparing a conjugate of theinvention which is useful for enhancing the uptake of nucleic acid intohigher eucaryotic cells, characterized in that a virus or a(poly)peptidic endosomolytic agent and a polyamine are chemicallycoupled.

The invention also relates to a process of preparing a conjugate of theinvention which is useful for enhancing the uptake of nucleic acid intohigher eucaryotic cells, comprising

a) modifying a virus or a virus component with biotin, and

b) binding the modified virus or modified virus component obtained instep a) to a streptavidin-coupled polyamine.

The invention also relates to a process for introducing nucleic acidinto higher eucaryotic cells, characterized in that the cells arecontacted with a composition according to the invention.

The invention also relates to a process for introducing nucleic acidinto liver cells in vivo, wherein the composition according to theinvention is administered to the liver via the bile duct.

The invention also relates to a process for producing a protein ofinterest in a higher eucaryotic cell, characterized in that the cellsare treated with a DNA complex of the invention, the nucleic acidcomprising a DNA sequence encoding the desired protein, the cells arecultivated under conditions suitable for expression of the protein, andthe protein is recovered.

The invention also relates to a pharmaceutical preparation comprising aDNA complex of the invention, wherein the nucleic acid istherapeutically active, and a pharmaceutically acceptable carrier.

The invention also relates to a transfection kit, comprising a carriermeans having in close confinement therein two or more container means,wherein a first container means contains a substance having an affinityfor nucleic acid, which substance is optionally coupled with aninternalizing factor for a higher eucaryotic cell; and a secondcontainer means contains an agent which has the ability per se of beinginternalized into the cells and of releasing the contents of theendosome into the cytoplasm.

The invention also relates to a transfection kit, comprising a carriermeans having in close confinement therein one or more container means,wherein a first container means contains a substance having an affinityfor nucleic acid, which substance is optionally coupled with aninternalizing factor for a higher eucaryotic cell, and wherein a secondcontainer means contains a substance having an affinity for a nucleicacid which is coupled to an agent which has the ability of beinginternalized into the cells as a component of a nucleic acid complex,and of releasing the contents of the endosome, in which the complex islocated after entering the cell, into the cytoplasm.

The invention also relates to a transfection kit, comprising a carriermeans having in close confinement therein one or more container means,wherein a first container means contains a biotin-modified endosomolyticagent and a second container means contains a streptavidin-modifiedsubstance having affinity for nucleic acid.

DESCRIPTION OF THE FIGURES

FIG. 1: Effect of adenovirus infection on gene transfer by means oftransferrin-polylysine conjugates.

FIG. 2: Conjugate-DNA-complex dosage effect.

FIG. 3: Enhancement of transferrin-polylysine mediated gene transfer byadenoviruses occurs by means of receptor-mediated endocytosis.

A) Effect on complexed DNA.

B) Effect on receptor-bound DNA.

C) Effect on gene transfer by means of transferrin-polylysineconjugates.

FIG. 4: Effect of adenovirus infection on gene transfer by means oftransferrin-polylysine conjugates in selected cell lines.

FIG. 5: Investigation into whether the enhancement of gene expression isbased on gene transfer or on transactivation.

A) Cell line K562.

B) Cell line K562 10/6 which constitutively expresses luciferase.

FIG. 6: Tetra-galactose peptide-polylysine conjugate.

FIG. 7: Transfection of HepG2 cells in the presence of adenovirus.

FIG. 8: Transfection of HepG2 cells in the presence of adenovirus.

FIG. 9: Transfection of TIB73 cells.

A) Comparison values with chloroquine.

B) In the presence of adenovirus.

FIG. 10: Transfection of T cells in the presence of adenovirus:

A) H9 cells.

B) Primary lymphocytes.

FIG. 11: UV-inactivation of adenoviruses:

A) Enhancement of gene transfer effect in HeLa cells by UV-inactivatedviruses.

B) Comparison of UV-inactivation with the gene transfer effect.

FIG. 12: Inactivation of adenoviruses by means of formaldehyde.

FIG. 13: Transfection of NIH3T3 cells in the presence of Moloney virus.

FIG. 14: Investigation into whether the gene transfer effect in thetransfection of NIH3T3 cells with transferrin-polylysine DNA complexescan be attributed to Moloney virus.

FIG. 15: Interactions between transferrin and its receptor play a partin the gene transfer effect of Moloney virus.

FIG. 16: Influence of pH on the gene transfer effect of retroviruses.

FIG. 17: Influenza-hemagglutinin peptide; liposome leakage assay.

FIG. 18: Transfection of K562-cells in the presence of influenzapeptide-polylysine conjugate p16pL.

FIG. 19: Transfection of HeLa cells in the presence of influenzapeptide-polylysine conjugate p16pL.

FIG. 20: Transfection of HeLa cells with transferrin-polylysineconjugates in the presence of influenza peptide-polylysine conjugatep41pL.

FIG. 21: In situ evidence of β-galactosidase expression aftertransfection of HeLa cells in the presence of adenovirus.

FIG. 22: Transfection of cells with a 48 kb cosmid in the presence ofadenovirus.

A: HeLa cells.

B: Neuroblastoma cells.

FIG. 23: Preparation of adenovirus-polylysine conjugates by chemicalcoupling.

FIG. 24: Transfection of KS62 cells by means of chemically coupledadenovirus conjugates.

FIG. 25: Transfection of HeLa cells by means of chemically coupledadenovirus conjugates.

FIG. 26: Binding of polylysine to adenovirus by means oftransglutaminase.

FIG. 27: Transfection of murine hepatocytes by means oftransglutaminase-coupled adenovirus conjugates.

FIG. 28: Increasing the efficiency of transfection bytransglutaminase-coupled adenovirus conjugates.

FIG. 29: Transfection of HeLa cells with biotin-streptavidin-coupledadenovirus conjugates.

FIG. 30: Transfection of K562 cells with biotin-streptavidin coupledadenovirus conjugates.

FIG. 31: Transfection of neuroblastoma cells with a 48 kb cosmid bymeans of biotin-streptavidin coupled adenovirus.

FIG. 32: Transfection of hepatocytes in the presence of chloroquine orin the presence of adenovirus.

FIG. 33: Transfection of K562 cells in the presence of variousendosomolytic agents.

FIG. 34: Comparison of transfection protocols at the cellular level withβ-galactosidase as a reporter gene in the presence of variousendosomolytic agents.

FIG. 35: Long term persistence of luciferase expression in confluent,non-dividing hepatocytes.

FIG. 36: Comparison of expression in HeLa cells transfected in thepresence of the chick embryo lethal orphan virus (CELO) in the free formand with CELO virus linked to polylysine via biotin-streptavidin.

FIG. 37: Transfection of myoblasts and myotubes withDNA/transferrinpolylysine complexes in the presence of free adenovirusand in the presence of biotin/streptavidin-coupled adenovirus.

FIG. 38: Delivery of DNA to mouse primary myoblast and myotube cultures.

FIG. 39: Comparative analysis of adenovirus d1312 and CELO virus in thetransfection of HeLa cells and C2C12 myoblasts.

FIG. 40: Improvement of CELO virus delivery to myoblasts using a lectinligand.

FIG. 41: Expression of a full length factor VIII cDNA in C2C12 myoblastand myotube cultures.

FIG. 42: Augmentation of DNA delivery by adenovirus proteins.

A) HeLa cells B) fibroblasts.

FIG. 43: Galactose-influenza peptide conjugates for DNA transfer intohepatocytes.

FIG. 44: Galactose-adenovirus conjugates for DNA transfer intohepatocytes.

FIG. 45: DNA transfer with transferrin-polylysine in the presence ofrhinovirus. A) free rhinovirus B) conjugated rhinovirus.

FIG. 46: Transfection of primary human melanoma cells with combinationcomplexes containing adenovirus conjugates.

FIG. 47: Transfection of primary human melanoma cells with combinationcomplexes comprising the low density lipoprotein.

FIG. 48: Liposome leakage assay of amphipathic peptides.

FIG. 49: Erythrocyte leakage assay of amphipathic peptides.

FIG. 50: Transfection of BNL CL.2 cells in the presence of amphipathicpeptides.

FIG. 51: Transfection of NIH3T3 cells in the presence of amphipathicpeptides.

FIG. 52: Expression of interferon alpha in HeLa cells transfected in thepresence of various endosomolytic agents.

FIG. 53: Transfection of B-lymphoblastoid cells with human-Ig polylysineconjugates and anti-human-Ig polylysine conjugates.

FIG. 54 The time course of heterologous gene expression in cotton ratairway epithelium transduced with humantransferrin-adenovirus-polylysine-DNA complexes.

FIG. 55: The results of the immunohistochemical staining ofconjugate-DNA complexes and parts thereof with human trachea.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The aim of the present invention was to improve the transfer of nucleicacid into higher eucaryotic cells. (The word “transfer” within the scopeof this invention means, apart from the introduction of the nucleic acidcomplexes into the cell through the cell membrane, the transport of thecomplexes or the nucleic acid released therefrom within the cell untilit reaches an appropriate site to be expressed). The higher eucaryoticcells are well known and do not include yeast. See Molecular Biology ofthe Gene, James D. Watson et al., the Benjamin/Cummings PublishingCompany, Inc., pp. 676-677 (1987).

A plurality of viruses effect their entry into the eucaryotic host bymeans of mechanisms which correspond in principle to the mechanism ofreceptor-mediated endocytosis. Virus infection based on this mechanismgenerally begins with the binding of virus particles to receptors on thecell membrane. After this, the virus is internalized into the cell. Thisinternalizing process follows a common route, corresponding to theentrance of physiological ligands or macromolecules into the cell: firstof all, the receptors on the cell surface arrange themselves in groups,and the membrane is inverted inwardly and forms a vesicle surrounded bya clathrin coating. After this vesicle has rid itself of its clathrincoat, acidification takes place inside it by means of a proton pumplocated in the membrane. This triggers the release of the virus from theendosome. Depending on whether the virus has a lipid coat or not, twotypes of virus release from the endosome were taken into account: in thecase of so-called “naked” viruses (e.g. adenovirus, poliovirus,rhinovirus) it was suggested that the low pH causes changes inconfiguration in virus proteins. This exposes hydrophobic domains whichare not accessible at the physiological pH. These domains thus acquirethe ability to interact with the endosome membrane and thereby cause therelease of the virus genome from the endosome into the cytoplasm. As forviruses with a lipid coat (e.g. vesicular stomatitis virus, SemlikiForest virus, influenza virus) it is presumed that the low pH modifiesthe structure or configuration of some virus proteins, thereby promotingthe fusion of the virus membrane with the endosome membrane. Viruseswhich penetrate into the cell by means of this mechanism have certainmolecular peculiarities which enable them to break up the endosomemembrane in order to gain entry into the cytoplasm.

Other viruses, e.g. the coated viruses Sendai, HIV and some strains ofMoloney leukaemia virus, or the uncoated viruses SV40 and polyoma, donot need a low pH for penetration into the cell; they can either bringabout fusion with the membrane directly on the surface of the cell(Sendai virus, possibly HIV) or they are capable of triggeringmechanisms for breaking up the cell membrane or passing through it. Itis assumed that the viruses which are independent of pH are also capableof using the endocytosis route (McClure et al., 1990).

When solving the problem of the invention, the starting premise was tomake use of the mechanism used by certain viruses to penetrate intoeucaryotic cells, in order to improve the transfer of nucleic acidcomplexes into the cell and thereby increase expression.

Attempts have been made to internalize proteins together with virusesinto the cell (Otero and Crasco, 1987). It was found that thepermeability achieved in the cell by the virus is used to delivermacromolecules. The procedures taking place would appear to be fluidphase uptake mechanisms.

Using epidermal growth factor, EGF, conjugated to a toxin, it was foundthat this natural ligand, which is taken up into the cell by endocytosisafter binding to its receptor, lands in the same endosome together withthe adenovirus, which is also taken up into the cell byreceptor-mediated endocytosis, and is released from this endosome, againtogether with the virus, into the cytosol (FitzGerald et al., 1983).

It was found with the present invention, surprisingly, that certainagents (e.g. viruses, virus components or other compounds), whichexhibit the characteristics of certain viruses with regard to theirmechanism to enter into eucaryotic cells, substantially increase therate of expression of a nucleic acid imported into the cell as part of acomplex. This finding was particularly surprising as the nucleic acidcomplexes taken up into the cell are very large.

The present invention thus relates to a composition for the transfectionof higher eucaryotic cells with a complex of nucleic acid and asubstance having an affinity for nucleic acid, which substance isoptionally coupled with an internalizing factor, characterized in thatit contains an agent which has the ability of being internalized intothe cells which are being transfected, either per se or as a componentof the nucleic acid complexes, and of releasing the contents of theendosomes, in which the complex is located after entering the cell, intothe cytoplasm.

This agent is hereinafter referred to as “endosomolytic agent”.

The ability of the endosomolytic agents to be taken up into the cellsand to release the contents of the endosomes, in which they are locatedafter entering the cell, into the cytoplasm, is hereinafter referred toas “uptake function”. This uptake function comprises the ability to beinternalized into the cell actively, via receptor-dependent endocytosismechanisms, or passively, via the liquid phase or as a constituent ofthe nucleic acid complex, and the ability to break up endosomes, whichis generally referred to as endosomolysis.

In one embodiment of the invention the endosomolytic agent is a virus.In another embodiment the endosomolytic agent is a virus component. Thevirus or virus component employed in these embodiments of the inventionis hereinafter referred to as “free” virus (component).

Within the scope of the present invention, the activity of an increasingdose of free adenovirus on the gene transfer capacity of a constantquantity of transferrin-polylysine conjugate in HeLa cells wasinvestigated, using the luciferase gene as reporter gene. Theaugmentation in gene transfer brought about by the addition of freeadenovirus reached a peak at 1×10⁴ virus particles per cell, a numberwhich corresponds to the approximate number of adenovirus receptors perHeLa cell. The augmentation, up to 2000-fold, of luciferase expressioncompared with the expression achieved with the transferrin-polylysineconjugates alone, corresponded to the higher dose of virus. In anotherseries of experiments, the capacity of limiting quantities ofconjugate-DNA complexes was investigated in the presence of a constantdosage of free adenovirus. It was found that the uptake of adenovirusesinto the cells augmented the gene transfer mediated bytransferrin-polylysine over a wide range of DNA dosages. The maximumintensity of gene expression achieved by means of the conjugate-DNAcomplexes corresponded to the intensity achieved with 100 times less DNAwhen adenoviruses were used to increase the efficiency of transfection.

The effect of adenoviral infection on gene transfer was examined forboth uncomplexed DNA and DNA that had been complexed with polylysine ortransferrin-polylysine conjugates (FIG. 3A). By this analysis,adenoviral infection did not significantly augment transfer of naked,uncompleted DNA during transfection. In marked contrast, transfer of DNAcomplexed to polylysine or transferrin-polylysine conjugates wasaugmented by adenoviral infection. This effect was, however, muchgreater for the transferrin-polylysine conjugates. Since the polycationportion of the conjugate molecule not only serves to attach transferrinto DNA, but also effects significant structural changes in the DNA(Compacting into toroidal structures; Wagner et al., 1991a; thedisclosure of which is fully incorporated by reference herein), theseexperiments could not initially differentiate whether the observedeffect was on the basis of enhanced fluid-phase transport of thepolycation-condensed DNA or virus-augmented delivery of receptor-boundconjugate-DNA complex. To distinguish between these possibilities,sequential binding experiments were performed (FIG. 3B). Binding oftransferrin-polylysine-DNA or polylysine-DNA complexes at lowtemperature without internalization allowed removal of excess complex inthe fluid phase prior to adenoviral infection (FitzGerald et at., 1983).When administered in this fashion, delivery of the receptor-boundtransferrin-polylysine-DNA complexes was significantly augmented by theaddition of adenoviral particles, whereas the polylysine-DNA complexeswere not. Thus, it is the entry of DNA by the receptor-mediatedendocytosis pathway which is specifically enhanced.

Next, analysis was made of the specific adenoviral function which bringsabout enhanced receptor-mediated gene transfer (FIG. 3C). Mild heattreatment of virions does not alter their ability to bind to target cellmembranes but does affect their capacity to disrupt endosomes afterinternalization (Defer et al., 1990). Thus, the distinct effects ofviral binding and viral entry could be separately evaluated. In thisanalysis, heat inactivation of the adenoviruses completely abolishedtheir ability to enhance receptor-mediated gene transfer. This suggeststhat it is the capacity of the adenoviruses to disrupt endosomes as partof their entry mechanism which specifically effects enhancement of genedelivery by transferrin-polylysine conjugates. The fact that areplication-defective virus strain could result in an increase in geneexpression confirms the assumption that this phenomenon is not due tothe replication function but due to the uptake function of the virion.

To rule out the possibility that the increase in gene expression can beascribed to possible transactivation of the imported gene by the virus,experiments were carried out with a cell line which constitutivelyexpresses the RSV-LTR luciferase gene: adenoviruses show no effects inthis cell line, whereas in the parental cell line into which the genehad been introduced by means of transferrin-polylysine conjugates, therewas a significant increase in gene expression. This finding demonstratesthat the adenovirus influences events which take place beforetranscription and that its enhancing effect on gene transfer thus actsat the gene transfer level and not at the gene expression level (FIG.5).

Investigations were also carried out within the scope of the inventionto find out what effect free adenovirus has on gene transfer by means oftransferrin-polylysine conjugates in selected cell lines. It was foundthat the presence of transferrin receptors on target cells is necessary,but not sufficient in every case, to permit gene transfer bytransferrin-polylysine conjugates. Cell-specific factors relating to thefate of endosome-internalized conjugate-DNA complexes appear to be anextremely important determining factor in the levels of gene transferachievable by this route. In this regard, selected cell lines wereexamined for both gene transfer by transferrin-polylysine conjugates andaugmentation of gene transfer by adenoviruses (FIG. 4). Cells of acystic fibrosis cell line (CFT1) showed moderate levels of luciferasegene expression after treatment with transferrin-polylysine-DNAcomplexes. This level of expression was significantly augmented bytreatment with the adenovirus d1312. In marked contrast, KB cellstreated with the transferrin-polylysine-DNA complexes exhibited levelsof luciferase gene expression barely above background levels, despitethe presence of transferrin receptors. Treatment with adenovirus d1312,however, resulted in readily detectable luciferase activities in thesecells. Treatment with adenoviruses on HeLa cells had a similar effect,although this effect was substantially stronger in these cells. SinceHeLa cells and KB cells possess approximately the same number ofreceptors for the adenovirus, the difference in augmentation of the genetransfer may reflect the number of transferrin receptors characteristicof each cell type. However, in marked contrast to these results, thecell lines WI-38 and MRC-5, which are known to support adenoviralinfection very poorly (Precious and Russell, 1985), showed very littleaugmentation with d1312 of the gene expression achieved by means of theconjugate-DNA complexes alone. Treatment with free virus, e.g.adenovirus, would therefore appear to augment gene transfer by means ofconjugate-DNA complexes in those cases where the gene transfer ispossible by receptor-mediated endocytosis, as in the case of CFT1 cells,and also in some instances where gene transfer by this route appears tobe ineffective, as for HeLa and KB cells. The level of augmentationachieved varies significantly among different target cells, suggestingthat this effect is a function of both the number of virus receptors,e.g. adenovirus receptors, of a certain cell type and also the number oftransferrin receptors.

In case of the use of free virus, the substance having an affinity fornucleic acid, preferably an organic polycation, is preferably conjugatedwith an internalizing factor. It has, however, been found, according tothe invention, that under certain circumstances DNA complexed only witha substance having an affinity for nucleic acid, i.e. withoutinternalizing factor, can be introduced into the cell in the presence offree virus. It was also found that, in some cell lines, the complexesconsisting of nucleic acid and a substance having an affinity fornucleic acid can be introduced through the fluid phase if theconcentration of the complexes is high enough. The experiments carriedout within the scope of the present invention and previous ones showedthat an essential element for the uptake capacity of the nucleic acidcomplexes is their compactness, which can be ascribed to the condensingof the nucleic acid by the substance having an affinity for nucleicacid. If the substance having an affinity for nucleic acid hassufficient capacity for binding to the cell surface in order to enterinto the cell together with the virus, as well as being able to renderthe complex substantially electroneutral and condense the nucleic acidinto a compact structure, there may not be a need to increase the entrycapacity by covalently binding an internalizing factor to the substancehaving an affinity for nucleic acid in order to transfer the complexinto the cell by receptor-mediated endocytosis. Many cells have arelatively high affinity for certain substances having an affinity fornucleic acid, so that the conjugates of nucleic acid and binding factorare taken up into the cell without the need for an internalizing factor.This is true, for example, of hepatocytes, which have been found withinthe scope of the present invention to take up DNA-polylysine complexes.

In a preferred embodiment of the invention, the endosomolytic agent is avirus which is bound to a substance having an affinity for nucleic acidand which has the ability to enter the cell as part of aconjugate/nucleic acid complex and release the contents of theendosomes, in which the complex is located after entering the cell, intothe cytoplasm.

In another preferred embodiment, the endosomolytic agent is a viruscomponent which is bound to a substance having an affinity for nucleicacid and which has the ability to enter the cell as part of aconjugate/nucleic acid complex and release the contents of theendosomes, in which the complex is located after entering the cell, intothe cytoplasm.

Viruses or virus components bound to the nucleic acid binding domain,irrespective of the type of binding, are hereinafter designated “viralconjugates”.

The viral conjugates, which are also subject of the present invention,contain the virus or virus component as an integral part of theirfunctional construct and combine the advantages of vector systems basedon internalizing factor conjugates with the advantages which the virusesbring into these systems.

Furthermore, the viral conjugates according to these embodiments of theinvention have the advantage that they circumvent the fundamentalrestriction inherent in the known molecular conjugate systems, in that,unlike the known conjugates employed for gene transfer byreceptor-mediated endocytosis, they have a specific mechanism whichenables them to be released from the cell vesicle system.

The vector system employing viral conjugates constitutes a fundamentalconceptual departure from the recombinant viral vectors, in that theforeign DNA which is to be transported is carried on the outside of thevirion. Consequently, the viral conjugates according to the preferredembodiments of the invention can transport very large gene constructsinto the cell, with no restrictions as to the sequence.

The suitability of a virus, which is to be employed as free or boundvirus or part of a virus as virus component within the scope of thepresent invention is defined by its uptake function as defined herein.Suitable viruses are, on the one hand, those which have the ability toenter into the cell by receptor-mediated endocytosis during transfectionof the cells with the nucleic acid complex and bring about theirrelease—and hence the release of the nucleic acid—from the endosome intothe cytoplasm. Such viruses are those disclosed herein as well as otherviruses capable of being taken up by a particular cell and causing therelease of the endosome contents into the cytoplasm.

For examples of viruses and higher eucaryotic cells into which they arecapable of penetrating, reference in made to Fields, B. N. and Knipe, D.M. (1990), the disclosure of which is fully incorporated by referenceherein. The susceptibility of a given cell line to transformation by avirus as a facilitator of conjugate entry as “free virus” is dependentupon the presence and number of target cell surface receptors for thevirus. With regard to the adenoviral cell surface receptor methods fordetermining its number on HeLa and KB cells are taught by Svensson,1990, and Defer, 1990, the disclosures of which are fully incorporatedby reference herein. It is thought that the receptor for the adenovirusis rather ubiquitously expressed.

Suitable viruses include, on the one hand, those which are able topenetrate into the cell by receptor-mediated endocytosis during thetransfection of the cells with the nucleic acid complex and to bringabout their release—and hence the release of the nucleic acid—from theendosome into the cytoplasm. Without wishing to be tied to this theory,this endosomolysis activity could, in the case of employing free virus,benefit the nucleic acid complexes transferred into the cell in so faras these complexes are conveyed together with the viruses from theendosomes into the cytoplasm, assuming that they arrive in the sameendosomes as the viruses on being internalized. When the complexescontain the virus in bound form they benefit from the virus'endosomolytic activity and are conveyed from the endosomes into thecytoplasm. This avoids the fusion between endosomes and lysosomes andconsequently the enzymatic degradation which normally takes place inthese cell organelles.

In particular, viruses which are suitable for the composition accordingto the invention and whose uptake function, occurring at the start ofinfection, occurs by receptor-mediated endocytosis, include on the onehand viruses without a lipid coat such as adenovirus, poliovirus,rhinovirus, and on the other hand the enveloped viruses vesicularstomatitis virus, Semliki Forest virus, influenza virus; pH-dependentstrains of Moloney virus are also suitable. Particularly preferredviruses which may be used in the practice of the invention includeAdenovirus subgroup C, type 5, Semliki Forest Virus, VesicularStomatitis Virus, Poliovirus, Rhinoviruses and Moloney Leukemia Virus.

The use of RNA viruses which have no reverse transcriptase, in thepresent invention has the advantage that transfection in the presence ofsuch a virus does not result in generation of viral DNA in thetransfected cell. In the present invention, Rhinovirus HRV2, arepresentative of the Picornavirus group, was shown to increaseexpression of a reporter gene. The efficacy of the Rhinovirus wasdemonstrated both in free form and in the form of virus conjugates.

Within the scope of the present invention, the term viruses—providedthat they are taken up into the cell and release the contents of theendosomes in which they arrive—includes in addition to the wild types,mutants which have lost certain functions of the wild type, other thantheir uptake function, especially their ability to replicate, as aresult of one or more mutations.

Mutants are produced by conventional mutagenesis processes by mutationsin virus-protein regions which are responsible for the replicativefunctions and which may be complemented by a packaging line. Theseinclude, e.g. in the case of adenovirus, ts-mutants (temperaturesensitive mutants), E1A- and E1B-mutants, mutants which exhibitmutations in MLP-driven genes (Berkner, 1988; the disclosure of which isfully incorporated by reference herein) and mutants which exhibitmutations in the regions of certain capsid proteins. Virus strains whichhave corresponding natural mutations are also suitable. The ability ofviruses to replicate can be investigated, for example, using plaqueassays known from the literature, in which cell cultures are coveredwith suspensions of various virus concentrations and the number of lysedcells which is visible by means of plaques is recorded (Dulbecco, 1980;the disclosure of which is fully incorporated by reference herein).

Other viruses which may be suitable for use within the scope of theinvention include so-called defective viruses, i.e. viruses which lackthe function necessary for autonomous virus replication in one or moregenes, for which they require helper viruses. Examples of this categoryare DI-particles (defective interfering particles) which are derivedfrom the infectious standard virus, have the same structural proteins asthe standard virus, have mutations and require the standard virus as ahelper virus for replication (Huang, 1987; Holland, 1990; thedisclosures of which are fully incorporated by reference herein).Examples of this group also include the satellite viruses (Holland,1990). Another group is the class of parvoviruses calledadeno-associated virus (Berns, K. I., 1990; the disclosure of which isfully incorporated by reference herein).

Since the entry cycles of many viruses are not completely characterized,it is likely that there will be other viruses that will exhibit theendosomolytic activity required for their suitability in the presentinvention.

Also suitable within the scope of this invention may be attenuated livevaccines (Ginsberg, 1980; the disclosure of which is fully incorporatedby reference herein) or vaccination strains.

The term viruses within the scope of the present invention also includesinactivated viruses, e.g. viruses inactivated by chemical treatment suchas treatment with formaldehyde, by UV-radiation, by chemical treatmentcombined with UV-radiation, e.g. psoralen/UV-radiation, bygamma-radiation or by neutron bombardment. Inactivated viruses, e.g.such as are also used for vaccines, may be prepared by standard methodsknown from the literature (Davis and Dulbecco, 1980, Hearst and Thiry,1977; the disclosures of which are fully incorporated by referenceherein) and tested for their suitability to increase the transfer of DNAcomplexes. In experiments carried out within the scope of the presentinvention, adenovirus preparations were inactivated using a conventionalUV sterilization lamp or with formaldehyde. It was surprisingly foundthat the degree of inactivation of the viruses was substantially greaterthan the reduction in the gene transfer effect, which was achieved whenadenovirus was added to the transfection medium. Experiments carried outby the inventors with preparations of psoralen(UV-inactivatedbiotinylated adenovirus, which was coupled with streptavidin-coupledpolylysine, also showed that as a result of the inactivation the virustiter decreased considerably more sharply than the gene transfercapacity. This is a clear indication that mechanisms which are connectedwith the normal replication in the active virus can be destroyed withoutdestroying the effect which is essential for gene transfer.

The term “virus components” denotes parts of viruses, e.g. the proteinpart freed from nucleic acid (the empty virus capsid, which may beproduced by recombinant methods, e.g. Ansardi et al., 1991; Urakawa etal., 1989; the disclosures of which are fully incorporated by referenceherein), proteins obtained by fractionation or peptides which have theendosomolytic functions of the intact virus. These virus components maybe produced synthetically, depending on their size either by peptidesynthesis or by recombinant methods. In the present invention adenovirusproteins conjugated via biotin/streptavidin to polylysine weredemonstrated to enhance gene transfer. Examples of fragments or proteinsfrom other than adenovirus, which are essential for internalization,include influenza virus hemagglutinin (HA). The N-terminal sequence ofthe influenza virus hemagglutinin HA2 subunit is responsible forreleasing the virus from the endosome. It has been shown that peptidesconsisting of 20 amino acids of this sequence are capable of fusinglipid membranes and partly breaking them open or destroying them(Wharton et al., 1988). In the present invention, authentic and modifiedinfluenza peptides were successfully employed in various embodiments.Another example are coat proteins of retroviruses, e.g. HIV gp41(Rafalski et al., 1990) or parts of these virus proteins.

The use of viruses which have the ability per se to enter cells and thusfunction as internalization factors, is but one aspect of the presentinvention.

Viruses or virus components which themselves do not bring the capacityto bind to the cell and enter into it, are preferably used as viralconjugates as defined above. Coupling to a DNA binding domain, e.g. apolycation, ensures that the virus (component) acquires a high affinityto DNA molecules and is thus complexed to it and transported into thecell as a component of the nucleic acid complex, which also contains aconjugate of internalizing factor and DNA binding domain. In addition tothe transfer effect thus achieved, binding of the virus (component) to anucleic acid binding domain may also result in an improvement of itsendosomolytic properties.

By choosing other internalization factors described herein, practicallyany higher eucaryotic cell may be transfected with the compositions ofthe present invention.

One can determine with a simple screening assay whether a given virus(component) has an uptake function and can be employed in the practiceof the invention. In this assay, e.g. for testing a virus for itsapplicability as free virus, the target cells are contacted with a DNAcomplex in the presence or absence of the virus. The amount of DNAcomplex released into the cytoplasm can then be easily determined bydetection of a marker gene product, e.g. luciferase. If the presence ofthe virus causes the DNA complex to be taken up and released into thecytoplasm at a greater level than without the virus, this may beattributed to the uptake function of the virus. It is also possible tocompare the level of DNA complex uptake with the test virus whencompared to another virus known to have a suitable uptake function, e.g.adenovirus subgroup C, type 5. Tests of this kind may also be applied toviral conjugates, additional parameters such as various internalizingfactor conjugates in varying amounts may be subject to such tests.Furthermore, a person skilled in the art can easily apply assays of thiskind, optionally in combination with other tests, e.g. liposome leakageassays, for testing virus components or other agents with potentialendosomolytic activity for their ability to enhance gene expression.

When intact viruses are used, tests are carried out, preferably parallelto the preliminary tests investigating the virus for its ability toaugment gene transfer, to see whether the virus is capable ofreplicating. The investigation for ability to replicate is carried outby using plaque assays (see above) or CPE assays or determination oflate gene expression in the case of cytopathic viruses or in the case ofviruses which significantly impair the growth of the host cells. Forother viruses, detection methods specific to the virus in question areused, e.g. the hemagglutination test or chemico-physical methods, e.g.using an electron microscope.

Within the scope of this invention, the preferred viruses, in particularthose which are applied as free viruses, are those which can be producedin a high titer, which are stable, have low pathogenicity in theirnative state and in which a targeted elimination of the ability toreplicate is possible, especially adenoviruses. If a specific cellpopulation is to be transfected, viruses which specifically infect thiscell population may be employed. If the transfection is intended totarget different cell types, viruses which are infectious for a widerange of cell types may be used.

The requirements on the compositions of free virus essentially are thatthe virus preparation should be of the greatest possible purity and thata stabilizing buffer should be used which is matched to the particularvirus.

In any case, for therapeutic use of the invention only those viruses orvirus components may be used in which the safety risks are minimized asfar as possible, particularly the risk of replication of the virus inthe target cell and recombination of virus DNA with host DNA.

Advantageously, the entry mechanism of viruses which infect animalsother than humans may be used to enhance the uptake and release of DNAinto higher eucaryotic cells, especially of humans, so long as the virusexhibits endosome disruption activity in the higher eucaryotic cells.Members of the adenovirus family have been isolated from avian species,from amphibians and from a variety of other animals. See, for example,Laver, W. G. et al., 1971;, Bragg, R. R. et al., 1991; Akopian, T. A. etal., 1991; Takase, K. et al., 1990; Khang, C. and Nagaraji, K. V., 1989;and Reece, R. L. et al., 1987; the disclosures of which are fullyincorporated by reference herein. Amphibian, avian, bovine, canine,murine, ovine, porcine, and simian adenoviruses, as well as humanadenoviruses, are available from the American Type Culture Collection,Rockville, Md. (See the American Type Culture Collection Catalogue ofAnimal Viruses and Antisera, Chlamydae and Rickettsiae, Sixth Edition,1990, C. Buck and G. Paulino eds., pp. 1-17).

One possible advantage of using a virus, e.g. an adenovirus, from adistant species might be a reduced toxicity in the target cells (e.g.the chicken or frog adenovirus would not be expected to replicate orinitiate early gene expression in mammalian cells), a reduced hazard tothe investigator preparing the distant adenovirus, compared to the humanadenovirus, and reduced interference in the target organism fromantibodies against human or murine adenovirus. The absence ofinterference by the human or murine antibodies is particularly importantwhen the viruses are employed in gene therapy in humans and mice.

The chicken adenovirus CELO (chick embryo lethal orphan virus) shows noreactivity to antibodies that recognize the major group epitopes of theadenoviruses infecting mammalian cells. Moreover, CELO may be grown inembryonated eggs to give high levels of virus (0.5 mg/egg; Laver et al.,1971). As shown in the Examples, CELO)-polylysine conjugates augment DNAdelivery to HeLa cells at levels comparable to the human adenovirusd1312. Thus, the use of CELO conjugates to augment DNA delivery holdsgreat promise in human gene therapy experiments.

Viruses of distant species are preferably used as constituents of viralconjugates in combination complexes, as herein defined.

In conjugates of the invention which contain a virus, binding of thevirus to the nucleic acid binding domain may be covalent ornon-covalent, e.g. a biotin-streptavidin bridge or a ionic binding incase the virus has areas on its surface proteins which are acidic andtherefore can bind to a polycation.

In experiments of the present invention, complexes were formed underconditions which allow ionic interaction between adenovirus andpolylysine before complexing with DNA. Control experiments wereconducted under conditions where polylysine is first neutralized withDNA and is therefore not free to bind the adenovirus. The complexes withionically bound adenovirus were superior in these experiments.

Examples for virus components in the endosomolytic conjugates of theinvention are the empty virus capsids or viral peptides. Binding of thevirus component to the nucleic acid binding domain may be covalent, e.g.by chemically coupling the viral peptide with polylysine, ornon-covalent, e.g. ionic in case the virus component has acid residuesto bind to a polycation.

The ratio of virus or virus component to the substance having affinityto nucleic acid may be varied. In the case of influenza haemagglutininpeptide-polylysine conjugate it was found in the present invention thatgene transfer can be augmented to a greater extent when the content ofviral peptide in the conjugates is higher.

In another aspect the present invention relates to methods of preparingthe viral conjugates according to the invention.

The conjugates of virus or virus component and substance having anaffinity for nucleic acid may be prepared (like the internalizingfactor-polycation conjugates) by binding the compounds or, if the viruscomponent and the nucleic acid binding domain are polypeptides, by therecombinant method; with regard to methods of preparation reference ismade to the disclosure of EP 388 758; the disclosure of which is fullyincorporated by reference herein.

Binding of virus or viral proteins or peptides, respectively, withpolyamine compounds by the chemical method can be effected in the mannerwhich is already known for the coupling of peptides and if necessary theindividual components may be provided with linker substances before thecoupling reaction (this measure is necessary if there is no functionalgroup available which is suitable for the coupling, e.g. a mercapto oralcohol group). The linker substances are bifunctional compounds whichare reacted first with functional groups of the individual components,after which the modified individual components are coupled.

Coupling may be carried out by means of

a) Disulphide bridges, which can be cleaved again under reducingconditions (e.g. when using succinimidyl-pyridyldithiopropionate (Junget al., 1981; the disclosure of which is fully incorporated by referenceherein).

b) Compounds which are substantially stable under biological conditions(e.g. thioethers by reacting maleimido linkers with sulfhydryl groups ofthe linker bound to the second component).

c) Bridges which are unstable under biological conditions, e.g. esterbonds, or acetal or ketal bonds which are unstable under slightly acidicconditions.

In experiments carried out within the scope of the present invention,endosomolytic influenza-hemagglutinin HA2-peptides were coupled withpolylysine by the chemical method usingsuccinimidylpyridyldithio-propionate (SPDP). It was shown that themodification of the peptide with polylysine increases the endosomolyticactivity. Transfection experiments showed that the efficiency of genetransfer mediated by transferrin-polylysine is substantially increasedif the influenza peptide-polylysine conjugates are present together withtransferrin-polylysine in the DNA complex.

Moreover, within the scope of the present invention, adenovirus wasbound to polylysine by various different methods. One way of conjugatingthe virus with polylysine was effected in a similar manner to theproduction of transferrin-polylysine conjugates (Wagner et al., 1990;the disclosure of which is fully incorporated by reference herein) aftermodification of the defective adenovirus d1312 using aheterobifunctional reagent. Unbound polylysine was removed bycentrifuging. The DNA binding capacity was demonstrated in a bindingexperiment using radioactively labelled DNA. (In K562 cells in theabsence of chloroquine, substantially higher gene transfer was foundwith complexes consisting of DNA, adenovirus-polylysine andtransferrin-polylysine, than with unmodified adenovirus which is notbound to the DNA. It was also found that significant gene expressionoccurred with only 0.0003 μg of DNA in5×10⁵ HeLa cells usingpolylysine-modified adenovirus.)

If the virus or virus component (or the additional internalizing factor,as e.g. in the case of transferrin) contains suitable carbohydratechains, they may be linked to the substance having an affinity fornucleic acid via one or more carbohydrate chains of the glycoprotein.

Another preferred method of preparing the viral conjugates of theinvention is by enzymatic coupling of the virus or virus component to asubstance having an affinity for nucleic acid, more particularly apolyamine, by means of a transglutaminase.

The category of transglutaminases comprises a number of differentenzymes which occur inter alia in the epidermis (epidermaltransglutaminase), in the blood (Factor XIII) and in the cells ofvarious tissues (tissue transglutaminase, e.g. liver transglutaminase)(Folk, 1985; the disclosure of which is fully incorporated by referenceherein). Transglutaminases catalyze the formation ofε-(γ-glutamyl)lysine bonds in the presence of Ca++ and with cleaving ofNH₃. The prerequisite for this is that corresponding glutamines andlysines should be present in proteins, capable of being reacted by theenzyme. Apart from the ε-amino group of lysine, (poly)amines such asethanolamine, putrescine, spermine or spermidine may also be used assubstrate (Clarke et al., 1959). At present it is not yet clear what thecritical factors are which determine whether a glutamine or lysine of aprotein or a polyamine can be reacted by the enzyme. What is known isthat polyamines can be bound by means of tansglutaminase to numerouscell proteins such as cytokeratins (Zatloukal et al., 1989), tubulin,cell membrane proteins and also surface proteins of influenza viruses(Iwanij, 1977).

Within the scope of the present invention it has been shown thatpolylysine can be coupled to adenoviruses by means of transglutaminase.It was found that coupling can be carried out in the presence ofglycerol. This has the advantage that a virus preparation, e.g. anadenovirus preparation which contains glycerol as stabilizing agent inthe buffer, can be used directly for coupling. Usingadenovirus-polylysine conjugates which were complexed with plasmid-DNAtogether with transferrin-polylysine conjugates, it was possible toachieve many times greater gene expression than withtransferrin-polylysine conjugates in the presence ofnon-polylysine-coupled adenovirus.

Another method of preparing the conjugates according to the inventionwhich is preferred within the scope of the invention consists incoupling the virus or virus component to the polycation via abiotin-protein bridge, preferably a biotin-streptavidin bridge.

The known strong association of biotin with streptavidin or avidin(Wilchek et al., 1988) was used for coupling adenovirus to polylysine bymodifying adenovirus with biotin and chemically conjugating streptavidinto polylysine in a similar manner to the product oftransferrin-polylysine conjugates (Wagner et al., 1990). Complexesconsisting of DNA and streptavidin-polylysine, to which thebiotin-modified virus is bound, and optionally non-covalently boundpolylysine, having a very high transfection efficiency, even at lowerconcentrations of DNA. Particularly efficient complexes are formed ifthe biotin-modified virus is first bound to streptavidin-polylysine andthe binding to DNA only occurs in a second step.

If desired, the binding to biotin may also be effected by means ofavidin.

It is also possible to establish the bond between the virus (component)and polylysine by biotinylating the virus, on the one hand, andconjugating an anti-biotin antibody with polylysine, on the other hand,and establishing the bond between the virus and the polylysine by meansof the biotin/antibody bond, using standard commercially availablepolyclonal or monoclonal anti-biotin antibodies.

Binding between the virus and polylysine may also be achieved bycoupling polylysine with a lectin which has an affinity for a virussurface glycoprotein, the bonding in such a conjugate being effected bymeans of the bond between the lectin and the glycoprotein. If the virusdoes not have any suitable carbohydrate side chains itself, it may besuitably modified.

A virus may also be bound to a substance having an affinity for nucleicacid by first being modified on the surface with an antigen alien to thevirus (e.g. digoxigenin DIG, obtainable from Boehringer Mannheim; orwith biotin) and establishing the connection between the modified virusand the substance having an affinity for nucleic acid via an antibodywhich binds to this antigen. The particular method which will be used toproduce the conjugates according to the invention depends on variouscriteria. Thus, for example, coupling by means of biotin is the leastspecific and therefore most widely applicable method, while thebiotin-mediated binding constitutes a very strong non-covalent bonding.The enzymatic reaction with transglutaminase has the advantage that itcan also be carried out on a very small scale. Chemical coupling isgenerally used when larger quantities of conjugate are to be synthesizedand this method is generally also the best when coupling virus proteinsor peptides. If inactivated viruses are used, the inactivation isgenerally carried out before the coupling, provided that the coupling isnot affected by the inactivation.

If a virus, e.g. adenovirus, or an endosomolytic component thereof, hasbinding domains accessible, e.g. acidic domains for binding to apolycation, binding of the virus (component) to the polycation may alsobe ionic. In this case, the positive charges of the polycation, which isoptionally conjugated with an internalizing factor, are partiallyneutralized by the acidic domain of the virus (component), the remainderof the positive charges will be essentially neutralized by the nucleicacid.

If the substance having an affinity for nucleic acid is an intercalatingsubstance, it is modified with a linker which is suitable for theparticular coupling of virus (component), e.g. for coupling withtransglutaminase it is modified with spermine or with a bifunctionalgroup competent for chemical coupling, e.g. an active ester.

The ratio of virus (component)/nucleic acid binding substances may vary,it is usually established empirically, e.g. by conjugating a constantamount of virus (component) with different amounts of polylysine andselecting the optimal conjugate for the transfection.

In another embodiment of the invention, the virus component, e.g. anendosomolytic viral peptide, may be modified in order to bind direct toDNA. To this end the peptide itself may contain a DNA binding domainwhich is obtainable by producing the peptide by means of peptidesynthesis and providing a stretch of positively charged amino acids,preferably by extending the peptide, most preferably at the C-terminus.

In another embodiment of the invention the endosomolytic agent is anon-viral, optionally synthetic peptide. A peptide of this type ispreferably contained in the composition according to the invention insuch a way that it is ionically bound to the substance with affinity tonucleic acid, e.g. to polylysine in case of DNA-internalizingfactor-polylysine complexes. Thereby incorporation of the endosomolyticpeptide into the nucleic acid complexes is accomplished by binding thepeptide via its acidic amino acid residues to the positively chargednucleic acid binding domain, preferably polylysine.

Depending on the chemical structure of the peptide, in particular withregard to its end group, binding to polylysine may also be accomplishedby the methods described herein for linking peptides to polylysine. Tothis end, if a naturally occurring peptide is employed, it may bemodified with a suitable terminal amino acid as a handle forconjugation.

Another way of incorporating non-viral endosomolytic peptides into thenucleic acid complexes is to provide them with sequences which bind toDNA. The location of such a sequence has to be such that it does notinterfere with the peptide's endosomolytic activity. Therefore, forexample, peptides whose N-terminus is responsible for this activity, areextended by DNA binding sequences at the C-terminus. Extensions of thiskind may be homologous or heterologous cationic oligopeptides, e.g. anoligo-lysine tail, or a natural DNA binding domain, e.g a peptidederived from a histone. Preferably these DNA binding sequences asintegral part of the endosomolytic peptide comprise approximately 10 to40 amino acids. This embodiment of the invention offers the possibilityof a higher ratio of endosomolytic sequence to DNA binding sequence thanin peptide conjugates which contain larger portions of polycations inorder to achieve a higher efficiency of the complexes.

The non-viral endosomolytic peptides should fulfil the followingrequirements:

With regard to endosomolytic activity the leakage of lipid membranesachieved by the peptide should preferably be higher at low pH (5-6) thanat pH 7. Furthermore, the disrupted areas of the membrane should belarge enough to allow passage of large DNA complexes (small pores arenot sufficient). In order to determine whether a peptide fulfills theserequirements, in vitro tests can be carried out by applying the peptidesin free or bound form and/or incorporated in a DNA complex. Such assaysmay comprise liposome leakage assays, erythrocyte leakage assays andcell culture experiments, in which augmentation of gene expression isdetermined. Tests of this type are described in the Examples. Theoptimal amount of peptide can be determined in preliminary titrations byassaying the resulting gene transfer efficiency. It has to be born inmind that efficiency of various peptides and optimal composition ofcomplex may depend on cell type.

Membrane disruptive peptides in general contain amphipathic sequences,namely a hydrophobic face that may interact with the lipid membrane, ahydrophilic face that stabilizes the aqueous phase at the membranedisruption site.

There are several examples of membane-disruptive peptides in nature,usually small peptides or peptide domains of large polypeptides. Suchpeptides may be classified according to their function in the naturalcontext, namely either in membrane disrupting peptides (e.g. peptides ofnaked viruses) and/or membrane fusing peptides (e.g. enveloped viruses).For the purpose of endosome disruption in the context of syntheticpeptides both classes of peptide sequences may be useful. Most of thenatural peptides are able to form amphipathic α-helices.

pH-specificity may be achieved by incorporation of acidic residues ontothe hydrophilic face of a putative amphipathic α-helix in such a waythat the helix can form only at acidic pH, but not at neutral pH wherecharge repulsion between the negatively charged acidic residues preventshelix formation. This property is also found with naturally occurringsequences (e.g. influenza HA-2 N-terminus).

A completely synthetic, rationally designed amphipathic peptide withpH-specific membrane-disruption properties has been described (Subbaraoet al., 1987; Parente et al., 1990; the disclosures of which are fullyincorporated by reference herein). This peptide (in free form) was shownto form only small pores in membranes, allowing only the release ofsmall compounds (Parente et al., 1990).

According to the embodiment of the invention which makes use ofnon-viral, optionally synthetic peptides, usually the following stepsare taken: a amphipathic peptide sequence is selected from the groups ofnaturally occurring or artificial peptides. Peptides of this kind areknown in the art, a survey of examples is given in Table 2. Ifnecessary, acidic residues (Glu, Asp) are introduced to make thepeptide's membrane disrupting activity more pH-specific (e.g. the doubleacid mutant of the influenza hemaggludinin peptide according to Example35, designated p50).

If necessary, acidic residues may also be introduced in order tofacilitate binding of the peptide to polylysine. One way to provide forsuch a polycation binding domain may be to introduce C-terminal acidicextensions, e.g. an oligo-Glu-tail.

Endosomolytic peptides suitable for the present invention may also beobtained by fusing naturally occurring and artificial sequences. In thepresent invention experiments were conducted with various peptides whichwere derived from the synthetic peptide GALA described by Parente etal., 1990. Some of the derivatives employed in the experiments of thepresent invention were obtained by combining the peptide GALA ormodifications thereof with sequences of the influenza peptide ormodifications thereof, e.g. the peptides designated EALA-Inf andEALA-P50 according to Example 35.

The length of the peptide sequence may be critical with regard to thestability of the amphipathic helix; an increase of stability of shortdomains derived from natural proteins, which lack the stabilizingprotein context, may be achieved by elongation of the helix.

In order to increase the endosomolytic activity of the peptides,homodimers, heterodimers or oligomers may be formed; it has been shownin the experiments of the present invention that a P50 dimer has a muchhigher activity than the monomer.

The present inventors have shown the effect of synthetic peptides on DNAuptake mediated by transferrin-polylysine conjugates. Various differentpeptides were synthesized, their liposome and erythrocyte leakagecapacity assayed and their effect on luciferase expression in TIB 73cells and in NIH3T3 cells tested.

In another embodiment of the invention, the endosomolytic agent may be anon-peptidic amphipathic substance. The requirements such a substancemust fulfil to be suitable for the present invention are essentially thesame as for the amphipathic peptides, namely ability to be incorporatedinto the nucleic acid complexes, pH specificity, etc.

In another aspect the invention relates to complexes which are taken upinto higher eucaryotic cells, containing nucleic acid and a conjugatewhich has the ability to form a complex with nucleic acid, forintroducing nucleic acid into higher eucaryotic cells. The complexes arecharacterized in that the conjugate consists of a substance having anaffinity for nucleic acid and an endosomolytic agent which is bound tothe substance having an affinity for nucleic acid and has the ability ofbeing internalized into the cell as part of a conjugate/nucleic acidcomplex and of releasing the contents of the endosomes, in which thecomplex is located after entering the cell, into the cytoplasm.

The nucleic acid complexes used within the scope of the invention arepreferably those wherein the nucleic acid is complexed with a substancehaving an affinity for nucleic acid in such a way that the complexes aresubstantially electroneutral.

In a preferred embodiment of the invention, the endosomolytic agent is avirus or a virus component covalently bound to a polycation.

Within the scope of the present invention, the endosomolytic conjugatesalso encompass—in addition to conjugates in which endosomolytic agentsare ionically bound to a DNA binding domain—endosomolytic agents whichbind to DNA direct, e.g. via their basic extension, although“conjugates” of this kind are strictly speaking not obtained byconjugation, i.e. by binding two compounds to each other. The functionof endosomolytc agents of this type as compounds of the compositionaccording to the invention is independent of whether they weresynthesized by conjugation of an endosomolytic agent and a DNA bindingdomain or whether a DNA binding domain was originally present in theendosomolytic agent.

In another preferred embodiment of the invention the complexes contain,in addition to the endosomolytic conjugate, another conjugate in which asubstance having an affinity for nucleic acid, in case of anendosomolytic polycation conjugate generally the same polycation as inthe conjugate, is conjugated to an internalizing factor having anaffinity for the target cell. This embodiment of the invention is usedparticularly when the target cell has no or few receptors for the virusemployed as part of the endosomolytic conjugate. Another application ofthis embodiment of the invention is when a virus component, e.g. anaturally occurring, optionally modified peptide, a non-viral,optionally synthetic endosomolytic peptide or a virus from a distantspecies are employed, which do not have the ability to penetrate bythemselves into the cells which are to be transfected. In the presenceof an additional internalizing factor-binding factor conjugate, theendosomolytic conjugates profit from the internalizing ability of thesecond conjugate, by being complexed to the nucleic acid together withthe second conjugate and being taken up into the cell as part of theresulting complex which in the following is referred to as “combinationcomplex” or “ternary complex”. Without being pinned down to this theory,the combination complexes are taken up by cells either by binding to thesurface receptor which is specific to the additional internalizingfactor or, e.g. in case a virus or virus component is used, by bindingto the virus receptor or by binding to both receptors and internalizedby receptor-mediated endocytosis. When the endosomolytic agents arereleased from the endosomes the DNA contained in the complexes is alsoreleased into the cytoplasm and thereby escapes the lysosomaldegradation.

In the experiments of the present invention, nearly all HeLa cells couldbe transfected with free adenovirus. The efficacy for hepatocytes couldbe still further improved when using ternary DNA complexes in which thereporter DNA is complexed to polylysine-transferrin conjugates andlinked to adenovirus. Here, co-localization of the endosomolytic virusand the ligand receptor complex in the endosome is guaranteed yieldingtransfection in virtually all cells for a variety of cells such asBNL.CL2 and HepG2 cells. In this instance, both viral and transferrinreceptors on the cell surface can act to capture the ternary DNAcomplexes. However, one can envisage also that DNA ternary complexes canbe internalized solely by the action of the cellular ligand/receptorassociation. Such a situation might be approximated in the experimentswhere ternary DNA complexes containing transferrin gained access to K562cells in the main via the transferrin receptor rather than theadenovirus receptor.

Unexpectedly, ternary complexes transferred DNA even when presented forDNA transfer at very low levels. Thus at an input of 30 pg DNA/3×10⁵cells, 1.8×10⁴ light units (resulting from expression of a luciferaseencoding plasmid) are obtained. At this input there are as little as 60DNA molecules and 1 PFU of virus per cell. This has to be compared tothe less efficient calcium precipitation protocol which uses 2×10⁵ DNAmolecules per cell (Molecular Cloning, Sambrook, J. et al. (Eds.), 2ndEdition, Vol. 3, pp. 16.39-16.40 (1989)). Thus, the present inventionrepresents a significant advance in the art since it allows for theefficient transformation of higher eucaryotic cells with very smallamounts of DNA.

The presence of viruses, virus components or non-viral endosomolyticagents in the DNA complexes as constituents of endosomolytic conjugateshas the following advantages:

1) Broader applicability of the gene transfer technology with nucleicacid complexes, since the endosomolytic agent itself, in particular incase a virus (component) is employed, may constitute the internalizingfactor or may also be complexed to the DNA together with anotherinternalizing factor (e.g. transferrin or asialofetuin etc.). In thisway it is possible to make use of the positive effect of the viruseseven for cells which do not have any receptor for the virus in question.

2) Improvement in the efficiency of gene transfer, since the binding ofthe endosomolytic conjugates to the DNA ensures that they are jointlytaken up into the cells. The coordinated uptake and release of virusesand DNA also gives rise to the possibility of a reduction in thequantity of DNA and viruses required for efficient gene transfer, whichis of particular importance for use in vivo.

The term “internalizing factor” for the purposes of the presentinvention refers to ligands or fragments thereof which, after binding tothe cell are internalized by endocytosis, preferably receptor-mediatedendocytosis, or factors, the binding or internalizing of which iscarried out by fusion with elements of the cell membrane.

Suitable internalizing factors include the ligands transferrin(Klausner, R. D. et al.,, 1983), conalbumin (Sennett, C. et al., 1981),asialoglycoproteins (such as asialotransferrin, asialorosomucoid orasialofetuin) (Ashwell, G. et al., 1982), lectins (Goldstein et al.,1980, Shardon, 1987) or substances which contain galactose and areinternalized by the asialoglycoprotein receptor; mannosylatedglycoproteins (Stahl, P. D. et al, 1987), lysosomal enzymes (Sly, W. etal., 1982), LDL (Goldstein, J. L. et al., 1982), modified LDL(Goldstein, J. L. et al., 1979), lipoproteins which are taken up intothe cells via receptors (apo B100/LDL); viral proteins such as the HIVprotein gp120; antibodies (Mellman, I. S. et al., 1984; Kuhn, L. C. etal., 1982, Abrahamson, D. R. et at., 1982), or fragments thereof againstcell surface antigens, e.g. anti-CD4, anti-CD7; cytokines such asinterleukin-1 (Mizel, S. B. et al., 1987), Interleukin 2 (Smith, K. A.et al., 1985), TNF (Imamure, K. et al., 1987), interferon (Anderson, P.et al., 1982), colony-stimulating factor (Walker, F. et al., 1987);factors and growth factors such as insulin (Marshall, S., 1985), EGF(Carpenter, G., 1984), platelet-derived growth factor (Heldin, C. -H. etal., 1982), transforming growth factor β (Massague, J. et al., 1986),nerve growth factor (Hosang, M. et al., 1987), insulin-like growthfactor I (Schalch, D. S. et al., 1986), LH, FSH, (Ascoli, M. et al.,1978), growth hormone (Hizuka, N. et al., 1981), prolactin (Posner, B.I. et al., 1982), glucagon (Asada-Kubota, M. et al., 1983), thyroidhormones (Cheng, S. -Y. et al., 1980); ∝-2-macroglobulin protease(Kaplan, J. et al., 1979); and “disarmed” toxins. Further examples areimmunoglobulins or fragments thereof as ligands for the Fc receptor oranti-immunoglobulin antibodies, which bind to SIgs (surfaceimmunoglobulins). The ligands may be of natural or synthetic origin.See, Trends Phamacol. Sci. 10:458-462 (1989); the disclosure of which isfully incorporated by reference herein, and the references citedtherein.

The following are essential requirements for the suitability of suchfactors according to the present invention,

a) that they can be internalized by the specific cell type into whichthe nucleic acid is to be introduced and their ability to beinternalized is not affected or only slightly affected if they areconjugated with the binding factor, and

b) that, within the scope of this property, they are capable of carryingnucleic acid “piggyback” into the cell by the route they use.

In the experiments carried out according to the invention, the widerange of uses of the invention regarding the internalizing factor, oradditional internalizing factor in the combination complexes,respectively, is demonstrated by means of human and mousetransferrin-polylysine (pL) conjugates, asialofetuin-pL conjugates,galactose-pL conjugates, wheat germ agglutinin conjugates, theT-cell-specific gp120pL and antiCD7-pL conjugates and by means of DNApolylysine complexes which do not contain any internalizing factor.Moreover, the performance of the virus conjugates according to theinvention was demonstrated by means of complexes of DNA andpolylysine-conjugated virus (or virus component) which contained noadditional internalizing factor-binding factor conjugate.

Specifically preliminary tests can be carried out to determine whether,in case the endosomolytic agent is a free virus, the use of aninternalizing factor, or in case the endosomolytic agent is a virus or avirus compound or a non-viral peptide which is part of an endosomolyticconjugate, an “additional” internalizing factor permits or improves theuptake of nucleic acid complexes. These tests comprise paralleltransfections with nucleic acid complexes, firstly without (additional)internalizing factor, e.g. in case of virus conjugates with complexesconsisting of nucleic acid and virus conjugate, and secondly withcomplexes in which the nucleic acid is complexed with another conjugateconsisting of an additional internalizing factor for which the targetcells have a receptor, and a substance having an affinity for nucleicacid.

If an internalizing factor is used, or if an additional internalizingfactor is used, i.e. a combination complex is applied, it is definedparticularly by the target cells, e.g. by specific surface antigens orreceptors specific to a cell type which thus permit the targetedtransfer of nucleic acid into this cell type.

Substances with an affinity for nucleic acid which may be used accordingto the invention include, for example, homologous organic polycationssuch as polylysine, polyarginine, polyornithine or heterologouspolycations having two or more different positively charged amino acids,these polycations possibly having different chain lengths, and alsonon-peptidic synthetic polycations such as polyethyleneimine. Othersubstances with an affinity for nucleic acid which are suitable arenatural DNA-binding proteins of a polycationic nature such as histonesor protamines or analogues or fragments thereof, as well as spermine orspermidines.

The length of the polycation is not critical, as long as the complexesare substantially electroneutral. The preferred range of polylysinechain lengths is from about 20 to about 1000 lysine monomers. However,for a given length of DNA, there is no critical length of thepolycation. Where the DNA consists of 6,000 bp and 12,000 negativecharges, the amount of polycation per mole DNA may be, e.g.:

60 moles of polylysine 200

30 moles of polylysine 400; or

120 moles of polylysine 100, etc.

One of ordinary skill in the art can select other combinations ofpolycation length and molar amount with no more than routineexperimentation.

Other suitable substances with an affinity for nucleic acid as part ofthe conjugates are intercalating substances such as ethidium dimers,acridine or intercalating peptides, containing tryptophan and/ortyrosine and/or phenylalanine.

As for the qualitative composition of the nucleic acid complexes,generally the nucleic acid to be transferred into the cell is determinedfirst. The nucleic acid is defined primarily by the biological effectwhich is to be achieved in the cell and, in the case of use for genetherapy, by the gene or gene section which is to be expressed, e.g. forthe purpose of replacing a defective gene, or by the target sequence ofa gene which is to be inhibited. The nucleic acids to be transportedinto the cell may be DNAs or RNAs, while there are no restrictionsimposed on the nucleotide sequence.

If the invention is applied on tumor cells in order to use them as acancer vaccine, the DNA to be introduced into the cell preferably codesfor an immune modulating substance, e.g. a cytokine like IL-2, IL-4, IFNgamma, TNF alpha. Combinations of cytokine encoding DNAs may beparticularly useful, e.g. IL-2 and IFN gamma. Another useful gene to beintroduced into tumor cells may be the multi drug resistance gene (mdr).In the present invention, transferrin-polylysine conjugates and lowdensity lipoprotein-polylysine conjugates were successfully employedtogether with the adenoviral conjugates to transfect tumor cells(melanoma cells). Depending on the individual application, preliminaryexperiments may be carried out with no more than routine experimentationin order to determine which ligand is best for a given tumor cell type.

It is also possible to introduce two or more different nucleic acidsequences into the cell, e.g. a plasmid containing cDNAs coding for twodifferent proteins under the control of suitable regulatory sequences ortwo different plasmid constructs containing different cDNAs.

Therapeutically effective inhibiting nucleic acids for transfer into thecells in order to inhibit specific gene sequences include geneconstructs from which antisense-RNA or ribozymes are transcribed.Furthermore, it is also possible to introduce oligonucleotides, e.g.antisense oligonucleotides, into the cell. Antisense oligonucleotidescomprise preferably 15 nucleotides or more. Optionally, theoligonucleotides may be multimerized. When ribozymes are to beintroduced into the cell, they are preferably introduced as part of agene construct which comprises stabilizing gene elements, e.g. tRNA geneelements. Gene constructs of this type are disclosed in EP A 0 387 775;the disclosure of which is fully incorporated by reference herein.

Apart from nucleic acid molecules which inhibit genes, e.g. viral genes,due to their complementarity, genes with a different mode of inhibitoryaction may be employed. Examples are genes coding for viral proteinswhich have so-called trans-dominant mutations (Herskowitz, 1987).Expression of the genes in the cell yields proteins which dominate thecorresponding wildtype protein and thus protect the cells, whichacquires “cellular immunity” by inhibiting viral replication.

Suitable are trans-dominant mutations of viral proteins which arerequired for replication and expression, e.g. Gag-, Tat and Rev mutantswhich were shown to inhibit HIV replication (Trono et al., 1989; Greenet al., 1989; Malim et al., 1989; the disclosure of which is fullyincorporated by reference herein).

Another mechanism of achieving intracellular immunity involvesexpression of RNA molecules containing the binding site for an essentialviral protein, e.g. so-called TAR decoys (Sullenger et al., 1990; thedisclosure of which is fully incorporated by reference herein).

Examples of genes which can be used in somatic gene therapy and whichcan be transferred into cells as components of gene constructs by meansof the present invention include factor VIII (hemophilia A) (see, e.g.Wood et al., 1984), factor IX (hemophilia B) (see, e.g. Kurachi, K. etal., (1982), adenosine deaminase (SCID) (see, e.g. Valerio, D. et al.,1984), ∝-1 antitrypsin (emphysema of the lungs) (see, e.g. Ciliberto, G.et al., 1985) or the cystic fibrosis transmembrane conductance regulatorgene (see, e.g. Riordan, J. R. et al., 1989; the disclosure of which isfully incorporated by reference herein). These examples do notconstitute a restriction of any kind.

As for the size of the nucleic acids, a wide range is possible; geneconstructs of about 0.15 kb (in case of a tRNA gene containing aribozyme) to about 50 kb or more may be transferred into the cells bymeans of the present invention; smaller nucleic acid molecules may beapplied as oligonucleotides.

It is clear that the widest possible applications are made possibleprecisely by the fact that the present invention is not subject to anylimitations on the gene sequence and the fact that very large geneconstructs may also be transferred by means of the invention.

Starting from the nucleic acid, the substance having an affinity fornucleic acid, preferably an organic polycationic substance, isdetermined, to ensure complexing of the nucleic acid, the obtainedcomplexes preferably being substantially electroneutral. If thecomplexes contain a conjugate of additional internalizing factor andsubstance having an affinity for nucleic acid, the polycation componentof both conjugates is taken into consideration with respect to theelectroneutrality aspect.

In the course of earlier inventions it had been found that the optimumtransfer of nucleic acid into the cell can be achieved if the ratio ofconjugate to nucleic acid is selected so that the internalizingfactor-polycation/nucleic acid complexes are substantiallyelectroneutral. It was found that the quantity of nucleic acid taken upinto the cell is not reduced if some of the transferrin-polycationconjugate is replaced by non-covalently bound polycation; in certaincases there may even be a substantial increase in DNA uptake (Wagner etal., 1991a). It had been observed that the DNA of the complexes ispresent in a form compressed into toroidal structures with a diameter of80 to 100 nm. The quantity of polycation is thus selected, with respectto the two parameters of electroneutrality and the achievement of acompact structure, while the quantity of polycation which results fromthe charging of the nucleic acid, with respect to achievingelectoneutrality, generally also guarantees compacting of the DNA.

Thus, in a further embodiment of the invention, the complexes alsocontain nucleic acid-binding substances in a non-covalently bound form,which may be identical to or different from the binding factor. In casethe endosomolytic agent is free virus, the complexes comprise nucleicacid and internalizing factor conjugate. In case an endosomolytic, e.g.a viral conjugate is employed, the nucleic acid is complexed with thisconjugate, optionally in concert with a conjugate of an additionalinternalizing factor. The choice of non-covalently bound “free”substances having an affinity for nucleic acid, in their nature andquantity, is also determined by the conjugate(s), particularly takingaccount of the binding factor contained in the conjugate: if, forexample, the binding factor is a substance which has no or limitedcapacity for DNA condensation, it is generally advisable, with a view toachieving efficient internalization of the complexes, to use substanceshaving an affinity for DNA which possess this property in a high degree.If the binding factor itself is a nucleic acid condensing substance andif it has already brought about compacting of the nucleic acidsufficient for effective internalization, it is advisable to use asubstance having an affinity for nucleic acid which brings about anincrease in expression by virtue of other mechanisms.

The suitable “free” substances having an affinity for nucleic acidaccording to the invention include compounds capable of condensingnucleic acid and/or of protecting them from undesirable degradation inthe cells, particularly the substances of a polycationic naturementioned hereinbefore. Another group of suitable substances comprisesthose which, by binding to the nucleic acid, bring about an improvementin the transcription/expression thereof, by improving the accessibilityof the nucleic acid for the expression machinery of the cell. An exampleof a substance of this kind is chromosomal non-histone protein HMG1,which has been found to possess the capacity to compact DNA and promotesexpression in the cell.

With regard to the complexes, when determining the molar ratios ofendosomolytic agent and/or internalizing factor/substance having anaffinity for nucleic acid/nucleic acid(s), care should be taken thatcomplexing of the nucleic acid(s) takes place, that the complex formedcan be bound to the cell and internalized, and that, either by itself orwith the aid of the endosomolytic agent is released from the endosomes.

The internalizing factor/binding factor/nucleic acid ratio dependsparticularly on the size of the polycation molecules and the number anddistribution of the positively charged groups, criteria which arematched to the size and structure of the nucleic acid(s) to betransported. Preferably, the molar ratio of internalizingfactor/substance having an affinity for a nucleic acid will range fromabout 10/1 to about 1/10.

After the construction and synthesis of the conjugates and determinationof the optimum ratio of conjugate:DNA for effective transfection, thequantity of the conjugate proportion which can be replaced, if desired,by free substance having an affinity for nucleic acid can be determinedby titration. If polycations are used both as the binding factor andalso as a free substance having an affinity for nucleic acid, thepolycations may be identical or different.

For the embodiment of the invention which employs viral conjugates amethod suitable for determining the ratio of the components contained inthe complexes may consist in first defining the gene construct which isto be introduced into the cells and, as described above, finding a virusor virus component which is suitable for the particular transfection.Then the virus or virus component is bound to a polycation and complexedwith the gene construct. Starting from a defined quantity of viralconjugate, titrations may be carried out by treating the target cellswith this (constant) quantity of conjugate and decreasing concentrationsof DNA, or vice versa. In this way the optimum ratio of DNA:virusconjugate is determined. If an additional internalizing factor is usedthe procedure may be, for example, to determine the optimum ratio ofvirus conjugate to internalizing factor conjugate starting from aconstant quantity of DNA by titration.

The complexes may be prepared by mixing together the components i)nucleic acid, ii) viral conjugate optionally iii) internalizingfactor/binding factor conjugate, and optionally iv) non-covalently boundsubstance having an affinity to nucleic acid, all of which may bepresent in the form of dilute solutions. If polycations are used as abinding factor and at the same time as “free” polycations, it isgenerally advisable first of all to prepare a mixture of conjugates with“free” polycations and then combine this mixture with DNA. The optimumratio of DNA to the conjugate(s) and polycations is determined bytitration experiments, i.e. in a series of transfection experimentsusing a constant amount of DNA and increasing amounts ofconjugate(s)/polycation mixture. The optimum ratio of conjugate(s):polycations in the mixture is obtained by routine experimentation or bycomparing the optimum proportions of the mixtures used in the titrationexperiments.

The DNA complexes may be prepared at physiological salt concentrations.Another possibility is to use high salt concentrations (about 2 M NaCl)and subsequent adjustment to physiological conditions by slow dilutionor dialysis.

The most suitable sequence for mixing the components nucleic acid,conjugate(s), possibly non-covalently bound substance with an affinityto nucleic acid is determined by prior experimentation. In some cases,it may prove advisable first to complex the nucleic acid with theconjugate(s) and then to add the “free” substance with an affinity fornucleic acid, e.g. the polycation, e.g. in the case of conjugates oftransferrin-ethidium dimer and polylysine.

In a preferred embodiment of the invention, the internalizing factor orthe additional internalizing factor, respectively, is transferrin andthe binding factor is a polycation. The term “transferrin” denotes boththe natural transferrins and also those transferrin modifications whichare bound by the receptor and transported into the cell.

The nucleic acid is taken up in the form of complexes in whichinternalizing factor-polycation conjugates are complexed with nucleicacid. When there is a content of a non-covalently bound substance withan affinity for nucleic acid, this is preferably a polycation. Thissecond polycation is identical to or different from the polycationcontained in the conjugate or in both conjugates.

In case of “combination complexes” the nucleic acid is internalized inthe form of complexes in which internalization factor conjugates on theone hand and endosomolytic conjugates on the other hand are complexedwith nucleic acid.

The conjugates of internalizing factor and polycation, which are usedtogether with free virus or together with the viral conjugates in thecombination complexes, may be prepared by a chemical method or, if thepolycation is a polypeptide, by a recombinant method; for methods ofpreparation, reference is made to the disclosure of EP 388 758; thedisclosure of which is fully incorporated by reference herein.

Preferably, within the scope of the present invention, conjugates areused in which the glycoprotein, e.g. transferrin, and the binding factorare connected to each other via one or more carbohydrate chains of theglycoprotein.

Unlike the conjugates prepared by conventional coupling methods,conjugates of this kind are free from modifications originating from thelinker substances used. In the case of glycoproteins which have only oneor a few carbohydrate groups suitable for coupling, e.g. transferrin,these conjugates also have the advantage that they are precisely definedin terms of their binding site for glycoproteins/binding factor.

A suitable method of preparing glycoprotein-polycation conjugates isdisclosed in German Patent application P 41 15 038.4; it was describedrecently by Wagner et al., 1991b; the disclosure of which is fullyincorporated by reference herein.

The quantity of endosomolytic agent used and the concentration thereofdepend on the particular transfection being undertaken. It is desirableto use the minimum quantity of virus or virus conjugate which isnecessary to ensure the internalization of the virus (conjugate) and thenucleic acid complex and release from the endosomes. The quantity ofvirus (conjugate) is matched to the particular cell type and theinfectivity of the virus for this type of cell must be taken intoconsideration above all. Another criterion is the particular conjugateof internalizing factor and binding factor, particularly with regard tothe internalizing factor, for which the target cell has a specificnumber of receptors. Moreover, the quantity of virus (conjugate) willdepend on the amount of DNA to be imported. Generally, a small amount ofvirus is sufficient for a stable transfection which requires only asmall amount of DNA, whereas a transient transfection, which requireslarger amounts of DNA, requires a larger quantity of virus. For aparticular application, preliminary tests are carried out with thetarget cells intended for transfection, possibly with a mixed cellpopulation, and the vector system envisaged for the transfection, inorder to determine the optimum virus concentration by titration, whilethe DNA used is conveniently a gene construct which largely coincideswith the one intended for concrete use, in terms of its size, andcontains a reporter gene for easier measurement of efficiency of genetransfer. Within the scope of the present invention, the luciferase andβ-galactosidase genes have been shown to be suitable reporter genes forsuch tests.

Another aspect of the invention relates to a process for introducingcomplexes of nucleic acid, a nucleic acid binding substance andoptionally an internalizing factor, into higher eucaryotic cells. Themethod is characterized in that the cells are brought into contact withan agent which has the ability of being internalized into the cellseither per se or as a component of the nucleic acid complexes and ofreleasing the contents of the endosomes, in which the nucleic acidcomplexes are located after entering the cell, into the cytoplasm.

In general, it is preferred to apply nucleic acid complex andendosomolytic agent simultaneously, but they may also be applied oneafter the other. In case of separate applications, the sequence ofapplication is not critical as long as the steps are carried out shortlyafter each other in order to guarantee that the components are ineffective simultaneous contact.

In case of using free virus in a separate preparation, simultaneousadministration of the preparation of free virus with the complexes maybe guaranteed by having the virus preparation as part of thetransfection medium which contains the nucleic acid complex.

In the case of simultaneous administration of free virus, the nucleicacid complexes and virus preparation are mixed together before beingadministered.

In a preferred embodiment, the endosomolytic agent is a component of acombination complex.

In order to increase gene expression, the compositions according to theinvention may also be administered repeatedly.

In a preferred embodiment, the cells are primary tumor cells. In aparticularly preferred embodiment the nucleic acid is a DNA whichcontains one or more sequences coding for an immune modulatingsubstance, preferably a cytokine.

In another embodiment the cells are myoblasts, preferably primarymyoblasts.

In another embodiment the cells are fibroblasts, preferably primaryfibroblasts.

In another embodiment the cells are hepatocytes, preferably primaryhepatocytes.

In another embodiment the cells are primary endothelial cells.

In another embodiment the cells are primary airway epithelial cells.

Table 1 shows the transfection success of the present inventionexemplified with various different cell types.

The composition of the invention was also investigated for transfectionof canine hemophilia B fibroblasts. Luciferase and β-galactosidase couldbe successfully expressed in these cells. Furthermore, the system wasused to deliver the 1.4 kb canine factor IX cDNA into fibroblasts from ahemophilic canine. In a sandwich ELISA, canine factor IX could bedetected 24 hours after transfection.

In certain cases, it is advisable to use a lysosomatropic substance inaddition to the endosomolytic agent, e.g. if the agent is aendosomolytic peptide conjugate or a retrovirus, the endosomolyticactivities of which are not strictly dependent on an acidic pH.

It is known that lysosomatropic substances inhibit the activity ofproteases and nucleases and may therefore inhibit the degradation ofnucleic acids (Luthmann and Mangusson, 1983; the disclosure of which isfully incorporated by reference herein). These substances includechloroquine, monensin, nigericin and methylamine. It has been shown thatmonensin brings about an increase in the expression of reporter genewhen a Moloney retrovirus is used.

The presence of chloroquine could be demonstrated to lead to expressionof a reporter gene, imported by transferrin-mediated DNA transfer invirtually 100% of K562 cells. BNL.CL2 or HepG2 hepatocytes did notrespond as well to chloroquine as did K562 cells but they could betransfected to a level of 5 -10% when exploiting the endosomolyticproperties of added replication defective or chemically inactivated freeadenovirus.

With the aid of the present invention, the advantages of the biologicalvectors are increased. As a result of the distribution of the receptorsthere is a tropism both for internalizing factor and for the virus. Bymatching these two components to the particular cell population, it ispossible to achieve a greater selectivity which is of particularimportance in the therapeutic application of this invention.

In another aspect the present invention relates to pharmaceuticalcompositions containing as active ingredient a complex oftherapeutically active nucleic acid, preferably as part of a geneconstruct, endosomolytic agent (optionally conjugated) and optionally aninternalizing factor conjugate, for administration to an animal, e.g. ahuman. Any inert pharmaceutically acceptable carrier may be used, suchas saline, or phosphate-buffered saline, or any such carrier in whichthe DNA complexes have suitable solubility properties for use in themethod of the present invention. Reference is made to Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (ed.)(1980) for methods of formulating pharmaceutical compositions.

The present invention offers the advantage of greatest possibleflexibility for application, inter alia as pharmaceutical composition.The composition of the invention may occur as a lyophilisate or in asuitable buffer in deep-frozen state. It may also be provided asready-to-use reagent in solution, preferably shipped cooled. Optionally,the components necessary for transfection, i.e. DNA, endosomolyticagent, optionally conjugated or ready for conjugation with a separateconjugation partner, DNA binding substance, optionally conjugated withan internalizing factor, optionally free polycation, may be present in asuitable buffer separate or partially separate as constituents of atransfection kit, which is also subject of the present invention. Thetransfection kit of the present invention comprises a carrier meanshaving in close confinement therein one or more container means such astubes, vials and the like, each of which contain the materials necessaryto carry out the transfection of a higher eucaryotic cell in accordancewith the present invention. In such a transfection kit, a firstcontainer means may contain one or more different DNAs, e.g. coding fordifferent antigens. A second container means may contain one or moredifferent internalizing factor conjugates that enable the use of thetransfection kit as a modular system. Whether the constituents aresupplied as a ready-to-use preparation or separately to be mixedimmediately before use, depends, apart from the specific application, onthe stability of the complexes, which can be determined routinely instability tests. In a preferred embodiment, a transglutaminase coupledadenovirus-polylysine conjugate, which has proven to be stable atstorage, is provided in one of the container means of a kit. In anotherpreferred embodiment, biotinylated adenovirus andstreptavidin-polylysine are provided in separate container means and aremixed before application. One of ordinary skill in the art can designnumerous different transfection kits to take advantage of theflexibility of the invention.

For therapeutic use, the composition may be administered systemically,preferably by intravenous route, as part of a pharmaceuticalcomposition. The target organs for this application may be, for example,the liver, spleen, lungs, bone marrow and tumors.

One example for local application is the lung tissue (use of thecomposition according to the invention as part of a pharmaceuticalcomposition in fluid form for instillation or as an aerosol forinhalation). In addition, the pharmaceutical compositions of theinvention may be administered by direct injection into the liver, themuscle tissue, into a tumor or by local administration in thegastro-intestinal tract. Another method of administration of thepharmaceutical composition is the application via the bile drainingsystem. This method of application allows direct access to hepatocytemembranes at the bile canaliculi, avoiding interaction of thecomposition with blood constituents.

Recently, the feasibility of using myoblasts (immature muscle cells) tocarry genes into the muscle fibres of mice was shown. Since themyoblasts were shown to secrete the gene product into the blood, thismethod may have a much wider application than treatment of geneticdefects of muscle cells like the defect involved in muscular dystrophy.Thus, engineered myoblasts may be used to deliver gene products whicheither act in the blood or are transported by the blood. The experimentsin the present invention have shown that both myoblast and myotubecultures, even primary ones, can be transfected with high efficiency.The most successful transfection media contained combination complexesof biotinylated adenovirus, transferrin-polylysine andstreptavidin-polylysine. Besides the reporter genes luciferase andβ-galactosidase, factor VIII was expressed in the muscle cells.Furthermore, the chicken adenovirus CELO was employed in combinationcomplexes containing wheat germ agglutinin as an additionalinternalizing factor.

Therapeutic application may also be ex vivo, in which the treated cells,e.g. bone marrow cells, hepatocytes or myoblasts, are returned to thebody (e.g., Ponder et at., 1991, Dhawan et al., 1991; the disclosures ofwhich are fully incorporated by reference herein). Another ex vivoapplication of the present invention concerns so-called “cancervaccines”. The principle of this therapeutic approach is to isolatetumor cells from a patient, transfect the cells with a cytokine-encodingDNA. The next step may involve inactivation of the cells, e.g. byirradiation, in such a way that they no longer replicate but stillexpress the cytokine. Then the genetically modified cells are applied tothe patient from which they have been isolated, as a vaccine. In theenvironment of the vaccination site, the secreted cytokines activate theimmune system, inter alia by activating cytotoxic T cells. Theseactivated cells are able to exert their effect in other parts of thebody and attack also non-treated tumor cells. Thus, the risk of tumorrecurrency and of developing metastasis are reduced. A protocol suitablefor the application of cancer vaccines for gene therapy was described byRosenberg et al., 1992; the disclosure of which is fully incorporated byreference herein. Instead of retroviral vectors suggested by Rosenberg,the gene transfer system of the present invention may be used. In theexperiments of the present invention primary melanoma cells weresuccessfully transfected with a reporter gene contained in combinationcomplexes of polylysine-coupled adenovirus and transferrin-polylysine.

The DNA complexes of the invention may be tested for in vivo efficacy inthe treatment of cystic fibrosis. The CF “knock-out” mouse modeldemonstrates prominent GI disease with relative sparing of the lung(Clarke et al., 1992). The DNA complexes of the present invention may betested to treat the pulmonary disease in this mouse model and to correctthe disease in the lower GI tract. Thus, in vivo gene transfer to GIepithelium in situ may be employed to achieve phenotypic correctionexperiments in the lower GI tract.

The present invention can also be used in assays for determining thehost immune response to a given antigen. Such assays are based on genetransfer to antigen-expressing cells.

Antigen-specific cytotoxic T lymphocytes (CTL) that kill infected cellsplay an important role in the host defence against viral infections ortumors. The interaction between T-cell and antigen-presenting cell (APC)is HLA (human lymphocytic antigens=MHC, major histocompatibilitymolecules)-restricted; to study CTL killing of cells expressing antigenin an in vitro CTL killing assay, one must present the antigen to theCTL in the correct HLA context, which usually means on an autologoustarget cell.

A CTL-killing assay may be performed as follows: APCs are transfectedwith an DNA construct containing an antigen encoding sequence. Antigenepitopes will be bound to MHC class I molecules and presented at thecell surface as a target for a specific CTL response. Thus, uponincubation with a sample of patient's serum, depending on the presenceof specific CTLs, the APCs will be lysed. Lysis is measured bymonitoring the release of e.g. radioactive chromium that wasincorporated into the APCs prior to the addition of the serum.Established protocols (Walker et al., 1989) use B-LCLs (B-lymphoblastoidcell lines) induced to express antigen genes by transfection withrecombinant vaccinia viruses. However, cells expressing antigenefficiently for about one day, die due to the lytic effect of vaccinia.

These difficulties can be overcome by CTL killing assays employing thegene transfer system of the invention for introducing antigen encodingDNA constructs, e.g. constructs encoding HIV or tumor antigens intofibroblasts to render them antigen expressing.

Primary fibroblasts are easy to obtain from biopsies, easy to grow, andhave been demonstrated to be transfectable with a particularly highefficiency (about 50 to about 70%) by means of the present invention.

Such assays are useful for identifying epitopes recognized by killercells in view of the design of vaccines. Furthermore, they can beadvantageously used in order to determine an individual's HLA restrictedimmune response against a given antigen.

Because a high level of expression of the transferred genes can beobtained in virtually all cells, the invention can be used to producerecombinant proteins. Here, there are no or few limitations as to thesequence and molecular weight of the transferred DNA, respectively.There is also a wide spectrum of cell types which are transfectable withthe DNA constructs of the present invention. Thus, nearly any cell typecan be used for the production of recombinant proteins which ensuresthat the recombinant protein is produced in a faithful and fullymodified post-translationally processed form guaranteeing highbiological activity of the product.

Gene transfer into cells may be accomplished as shown for luciferase andfor interferon alpha, practically any gene construct that gives rise toa desired protein product can be delivered. The desired protein productcan be recovered from the transfected cell culture (either the cellsupernatant or an appropriate cell homogenate, according to the protocolfor the particular protein product), 24 hours to one week or more afterthe transfection.

The application of the gene transfer system according to the presentinvention for the production of recombinant proteins has the followingadvantages:

1) Due to the high transfection efficiency (more than 90% of thetransfected cells can express the delivered gene at high levels), nopreselection of positively transfected cells is required and there is noneed for establishing stable cell lines. Small scale cell culture can besufficient to produce useful quantities of protein.

2) Large gene constructs may be delivered. Up to 48 kb have beensuccessfully delivered thus far.

3) The gene expression can be performed in cells that guarantee theappropriate post-translational processing and modification (e.g. vitaminK-dependent carboxylation of clotting factors, see Armentano, et at.,1990, or cell type specific glycosylation).

4) A broader selection of target cell types is made available for geneexpression using this method.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting inany way. All patents and publications cited herein are incorporated byreference herein in their entirety.

EXAMPLES

In the Examples which follow, illustrating the present invention, thefollowing materials and methods were used unless otherwise specified:

Preparation of transferrin-polylysine/DNA complexes

a) Human transferrin-polylysine conjugates

The method described by Wagner et al., 1991b, was used, in whichpolylysine is coupled to the carbohydrate side chains of transferrin.

A solution of 280 mg (3.5 μmol) of human transferrin (iron-free, Sigma)in 6 ml of 30 mM sodium acetate buffer, pH 5, was cooled to 0° C. and750 μl of 30 mM sodium acetate buffer pH 5 containing 11 mg (51 μmol) ofsodium periodate were added. The mixture was left to stand in the darkin an ice bath for 90 minutes.

In order to remove the low molecular products, gel filtration wascarried out, (Sephadex G-25, Pharmacia), yielding a solution whichcontained about 250 mg of oxidized transferrin (measured by ninhydrinassay). (In order to reveal the oxidized form which contains aldehydesand gives a color reaction when stained with anisaldehyde, the sampleswere added dropwise to a thin layer plate of silica gel and dried andthe plates were dipped into p-anisaldehyde/sulfuric acid/ethanol(1/1/18), dried and heated.) The modified transferrin solution was addedquickly (within 10 to 15 minutes) to a solution containing 1.5 μmol offluorescein-labelled poly(L)lysine with an average chain length of 190lysine monomers in 4.5 ml of 100 mM sodium acetate, pH 5. The pH of thesolution was adjusted to pH 7.5 by the addition of 1 M sodiumbicarbonate buffer. At intervals of 1 hour, 4 batches of 28.5 mg (450μmol) of sodium cyanoborohydride were added to the mixture. After 17hours, 2 ml of 5 M sodium chloride were added to adjust the solution toa total concentration of 0.75 M. The reaction mixture was loaded on acation exchange column (Pharmacia Mono S HR 10/10) and eluted with asalt gradient of 0.75 M to 2.5 M sodium chloride with a constant contentof 25 mM HEPES, pH 7.3. The high salt concentration when loading thecolumn and at the beginning of the gradient was essential for obtainingthe polycation conjugates. Some transferrin (about 30%) together with aweak fluorescence activity was eluted in the flow through; the majorityof fluorescence-labelled conjugate was eluted at a salt concentration ofbetween 1.35 M and 1.9 M and was pooled in 3 fractions. These fractions(in the sequence in which they were eluted) yielded, after two lots ofdialysis against 2 1 25 mM HEPES pH 7.3, a fraction A (TfpL190A)containing 45 mg (0.56 μmol) of transferrin, modified with 366 nmol ofpolylysine, a fraction B (TfpL190B) containing 72 mg (0.90 μmol)transferrin, modified with 557 nmol polylysine and a fractionC(TfpL190C), containing 7 mg (85 nmol) transferrin, modified with 225nmol polylysine. If they were not used immediately, the transferrinconjugates were flash-frozen in liquid nitrogen and stored at −20° C. iniron-free form. Before the incorporation of iron, samples (0.5 to 1 mg)were adjusted to a physiological salt concentration (150 mM) with sodiumchloride. The iron was incorporated by adding 4 μl of 10 mM iron (111)citrate buffer (containing 200 mM citrate, adjusted to a pH of 7.8 bythe addition of sodium bicarbonate) per mg of transferrin content. Theconjugates containing iron were divided up into small aliquots beforebeing used for DNA complexing, then flash frozen in liquid nitrogen ordry ice/ethanol and stored at −200° C. (this procedure proved advisableonce it was found that repeated thawing and freezing causes theconjugates to lose activity.)

b) Murine transferrin polylysine conjugates

A similar method was used as for human transferrin, in that coupling waseffected by means of the carbohydrate side chains. Conjugates of 15.5nmol murine transferrin and 13 nmol pL290 were obtained from 4.1 mg (51nmol) of murine transferrin and 2.1 mg (34 nmol) of pL 290.

Plasmid-DNA

a) pRSVL-DNA

The DNA plasmid pRSVL (containing the Pholinus pyralis luciferase geneunder the control of the Rous Sarcoma Virus LTR Enhancer/Promoter(Uchida et al., 1977, De Wet et al., 1987; the disclosures of which arefully incorporated by reference herein), was prepared using the Triton-xLysis standard method (Maniatis), followed by CsCl/EtBr equilibriumdensity gradient centrifugation, decolorizing with butanol-1 anddialysis against 10 mM Tris/HCl pH 7.5, 1 mM EDTA). For complexformation, in general, 6 μg of the DNA plasmid material in 350 μl HBS(150 mM NaCl, 20 mM HEPES, pH 7.3) were mixed with 12 μg oftransferrin-polylysine conjugate in 150 μl HBS, 30 minutes before addingto the cells.

b) pCMVL-DNA

The plasmid pCMVL (reporter gene construct containing the Photinuspyralis luciferase gene under the control of the cytomegaloviruspromoter) was prepared by removing the BamHI-Insert of the plasmidpSTCX556 (Severne et al., 1988; the disclosure of which is fullyincorporated by reference herein), the plasmid was treated with Klenowfragment and the HindIII/Ssp1 and Klenow-treated fragment from theplasmid pRSVL which contains the sequence coding for luciferase wasinserted. pCMVβ gal was described by Macgregor and Caskey (1989); thedisclosure of which is fully incorporated by reference herein. DNApreparation was carried out analogously to pRSVL.

Production of Virus Preparations

a) Adenovirus preparations

The adenovirus strain d1312 described by Jones and Shenk, 1979, having adeletion in the Ela region was used. Replication of the virus wascarried out in the Ela-trans-complementing cell line 293, and thepurification was carried out on a large scale as described by Davidsonand Hassell, 1987; the disclosure of which is fully incorporated byreference herein. The purified virus was taken up in storage buffer (100mM Tris, pH 8.0, 100 mM NaCl, 0.1% BSA, 50% glycerol) or in HBS/40%glycerol and aliquots were stored at −70° C. The virion concentrationwas determined by UV-spectrophotometric analysis of the extractedgenomic viral DNA (Formula: one optical density unit (OD, A₂₆₀)corresponds to 10¹² viral particles/ml; (Chardonnet and Dales, 1970)).

b) Retrovirus-Preparation

The Moloney murine leukaemia retrovirus N2 was packaged in an ecotropicpackaging line (Keller et al., 1985, Armentano et al., 1987; thedisclosures of which are fully incorporated by reference herein).Supernatants from virus expressing cells were collected, flash frozen inliquid nitrogen and stored at −20° C. The supernatants used in theExamples had a titer of approximately 10⁶ cfu/ml, as measured byneomycin-resistance colony formation with NIH3T3 cells. For the virusconcentration experiments, the supernatants were passed through a 300 kDexclusion membrane (FILTRON) in an AMICON stirred cell concentratorunder nitrogen pressure. Normally, 10 to 30 ml of supernatant wereconcentrated tenfold by this method.

Cells and Media

HeLa cells were cultivated in DMEM-Medium, supplemented with 5%heat-inactivated fetal calf serum (FCS), penicillin in amounts of 100I.U./ml, streptomycin (100 μg/ml) and 2 mM glutamine. WI-38, MRC-5, andKB cells were cultivated in EMEM-medium (Eagle's modified essentialmedium), supplemented with 10% heat inactivated FCS, antibiotics such asDMEM medium, 10 mM non essential amino acids and 2 mM glutamine. CFT1, arespiratory cystic fibrosis epithelial cell line (prepared by the methoddescribed by Yankaskas et al., 1991; the disclosure of which is fullyincorporated by reference herein; the CFT1 cell line is characterized inthat it is homozygous for the δF508 deletion CF-mutation) was cultivatedin F12-7X-medium (Willumsen et al., 1989). For the gene transferexperiments the cells were cultivated in 6 cm cell culture plates untilthey were about 50% confluent (5×10⁵ cells). The medium was removed and1 ml of DMEM or EMEM/2% FCS medium was added. Then the conjugate-DNAcomplexes were added, followed immediately by the adenovirus d1312(0.05-3.2×10⁴ particles per cell) or a comparable volume of virusstorage buffer (1-80 μl). The plates were returned to the incubator forone hour (5% CO₂, 37° C.), then 3 ml of complete medium were added.After a further 24 hours' incubation the cells were harvested in orderto measure the luciferase gene expression. In the case of the CFT1, thecells were cultivated for 4 hours in F12-7X medium without humantransferrin before the gene transfer experiments.

The following cell lines were obtained from ATCC, obtainable under theCatalogue Numbers given; HeLa cells: CCL 2, K562 cells: CCL 243, HepG2cells: HB 8065, TIB-73-cells: TIB 73 (BNL CL.2), NIH3T3 cells: CRL 1658,293 cells: CRL 1573, KB cells: CCL 17, WI-38 cells: CCL 75, MRC 5 cells:CCL 171. H9 cells were obtained from the AIDS Research and ReferenceReagent Program, U.S. Department of Health and Human Services, CatalogueNumber 87.

Primary lymphocytes were obtained by taking up a 25 ml sample ofumbilical cord blood in test tubes containing EDTA. Aliquots wereunderlayed with 4.5 ml of Ficoll-hypaque (Pharmacia) and centrifuged for15 minutes at 2,500 rpm. The brownish layer between the upper plasmalayer and the clear Ficoll layer was removed (about 10 ml). 40 ml ofIMDM plus 10% FCS was added, the sample was centrifuged at 1200 rpm for15 minutes and the cell pellet was suspended in 50 ml of fresh IMDM plus10% FCS (the cell density was about 2×10⁶ cells/ml). A 250 μl aliquot ofphytohaemagglutinin PHA P, DIFCO) was added, the culture was incubatedfor 48 hours at 37° C. and 5% CO₂, the recombinant IL-2 (BMB) was added(concentration: 20 units per ml). The cells were then split 1:3 withIMDM/20% FCS, 2 units/ml IL2. Aliquots of the cells were deep frozen inliquid nitrogen in FCS plus 5% DMSO. Before use, the cells were grown inIMDM plus 20% FCS plus 2 units ml/IL-2.

For the sequential binding investigations HeLa cells were equilibratedat 4° C. in 1 ml DMEM, supplemented with 2% FCS. The conjugate-DNAcomplexes were added as in the other tests and the plates were incubatedfor 2 hours at 4° C. Then the plates were exhaustively washed with icecold DMEM/2% FCS, then 2 ml of this medium were added. Adenovirus d1312or virus buffer was then added, the cells were left to warn up slowly,before being placed in the incubator for a further 24 hours. After thisincubation, the cells were harvested and investigated for luciferasegene expression.

Luciferase Assay

The preparation of cell extracts, standardization of the protein contentand determination of the luciferase activity were carried out asdescribed by Zenke et al., 1990, Cotten et al., 1990, and in EP 388 758;the disclosures of which are fully incorporated by reference herein.

Example 1 Determination of the Effect of the Adenovirus Treatment onGene Transfer by Transferrin-Polylysine Conjugates

First of all, the effect of an increase in dosage of virus on theability of a defined amount of conjugate-DNA complex to achieve genetransfer was investigated. For the complex formation, 6 μg of theplasmid pRSVL were mixed with 12 μg of human transferrin-polylysineconjugate (hTfpL190B). The conjugate-DNA complex plus various amounts ofthe adenovirus d1312 (0.05-3.2×10⁴ virus particles per cell) were addedto the HeLa cells. The results of this analysis are shown in FIG. 1. Theluciferase activity is expressed in light units of 50 μg of total cellprotein. According to this analysis, increasing amounts of addedadenovirus resulted in corresponding increases in gene transfer. Thefigure shows the averages from 2 to 4 separate experiments; the barsindicate standard deviation.

Example 2 Conjugate-DNA Complex Dosage Effect

Logarithmic dilutions of conjugate-DNA complexes prepared as in Example1, were added to HeLa cells either with or without the addition of aconstant dosage of adenovirus d1312 (1×10⁴ viral particles per cell).The luciferase activity was determined as in Example 1. The results areshown in FIG. 2.

Example 3 Enhancement of the Gene Transfer Effected by TransferrinPolylysine by Means of Adenovirus Occurs through Receptor-mediatedEndocytosis

a) Effect of adenovirus treatment on the transfer of the complexed DNA

The following components were used for transfection:

6 μg pRSVL-DNA without transferrin-polylysine conjugate (DNA); 6 μgpRSVL-DNA plus 6 μg of non-conjugated polylysine 270 (DNA+pL); 6 μg ofpRSVL-DNA plus 12 μg of transferrin-polylysine conjugates used inprevious examples (DNA+hTfpL190B). These transfection materials wereadded to the HeLa cells with or without adenovirus d1312 (dl1312) (1×10⁴viral particles per cell). The preparation of the cell extracts,standardization for total protein and determination of the luciferaseactivity were carried out as in the previous examples. The results ofthe tests are shown in FIG. 3A.

b) Effect of adenovirus treatment on the transfer of receptor-bound DNA

Conjugate-DNA complexes (DNA+hTfpL190B) or polylysine-DNA complexes(DNA+pL) were bound to HeLa without being internalized, by incubating at4° C. Non-bound complex was removed before the addition of adenovirusd1312 (d1312) (1×10⁴ viral particles per cell) or a comparable buffervolume. Subsequent incubation was carried out at 37° C. in order topermit internalization of the bound DNA complexes and adenoviruses. Theluciferase activity was determined as described (FIG. 3B).

c) Effect of adenovirus treatment of gene transfer bytransferrin-polylysine conjugates

Conjugate-DNA complexes containing 6 μg pRSVL-DNA plus 12 μgtransferrin-polylysine (DNA+hTfpL190B) were added to the HeLa cells with×10⁴ adenovirus particles (d1312) per cell or a comparable quantity ofheat-inactivated adenovirus d1312 (d1312 h.i.). Heat inactivation wascarried out by incubating for 30 minutes at 45° C. (Defer et al., 1990).FIG. 3C shows the luciferase activity.

Example 4 Effect of Adenovirus Treatment on Gene Transfer byTransferrin-polylysine Conjugates in Selected Cell Lines

Conjugate-DNA complexes (6 μg pRSVL+12 μg hTfpL190B) were added to cellsof the cell lines CFT1, KB, HeLa, W138 and MRC5 with or withoutadenovirus d1312 (1×10⁴ virus particles per cell). The efficiency ofgene transfer for the various cell lines was determined as in theprevious examples by luciferase assay (FIG. 4).

Example 5 Enhancement of Luciferase Gene Expression Functions at theLevel of Gene Transfer, Not at the Level of Transactivation

A cell line designated K562 10/6 constitutively expressing luciferasewas prepared by transfecting cells with a plasmid which contained anRSV-luciferase gene fragment (an Apa1/Pvu1 fragment of pRSVL (De Wet etal., 1987); the disclosure of which is fully incorporated by referenceherein), cloned into the Cla1 site of the pUCμ Locus (Collis et al.,1990; the disclosure of which is fully incorporated by referenceherein). This plasmid was complexed with a transferrin-polylysineconjugate and K562 cells were transfected with these complexes, usingthe method described by Cotten et al., 1990; the disclosure of which isfully incorporated by reference herein. Since the pUCμ Locus plasmidcontains a neomycin resistance gene it was possible to select forluciferase-expressing clones on the basis of neomycin resistance. Forthe further experiments, clone K562 10/6 was selected.

Aliquots of the parental cell line K562 (in 200 μl RPMI 1640 plus 2%FCS; 500,000 cells per sample) were treated either with 12 μg TfpL plus6 μg pRSVL or with 4 μg pL 90 plus 6 μg pRSVL, in 500 μl HBS in eithercase. The quantities of adenovirus d1312 specified (FIG. 5) were allowedto act on the cells for 1.5 hours at 37° C., after which 2 ml of RPMIand 10% FCS were added. Then incubation was continued at 37° C. for afurther 24 hours and the cells were then prepared for measurement forthe luciferase activity. It was found that incubation with adenovirusresults in a significant increase in the luciferase activity (FIG. 5A).This applies both to the TfpL complexes (2000 light units as against25,000 light units) and also to the pL 90 complexes (0 as against1.9×10⁶ light units). This shows that the K562 cell line has thecapacity to internalize pRSVL polylysine complexes and that thisinternalization, measured by luciferase expression, is significantlyincreased by the presence of adenovirus.

Analogous tests were carried out with the K561 10/6 cells whichconstitutively express the RSVL luciferase gene, and similar amounts ofadenovirus d1312 were used. Aliquots of 500,000 cells (in 200 μl RPMIplus 2% FCS) were incubated at 37° C. for 1.5 hours with the quantitiesof adenovirus d1312 specified in FIG. 5B. Then, as in the parental cellline RPMI plus 10% FCS was added, incubation was continued for a further24 hours and the luciferase activity was determined. As shown in FIG.5B, the treatment of these cells with the adenovirus does not have adetectable effect on the luciferase activity; the control values are inthe same range as the values for the virus treated samples.

Example 6 Transfection of Liver Cells with Asialofetuin-polylysineConjugates (AFpL) or with Tetra-galactose Peptide-pL Conjugates (gal4pL) in the Presence of Adenovirus

a) Preparation of the lactosylated peptide

3.5 mg (1.92 μmol) of the branched peptideLys-(N_(ε)-Lys)Lys-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Cys, preparedby the Fmoc method using an Applied Biosystems 431A Peptide Synthesizer,containing a dithiopyridine group for Cys, were treated with a solutionof 7.85 mg of lactose in 40 μl of 10 mM aqueous sodium acetate pH 5 at37° C. To the solution were added four aliquots of 0.6 mg (10 μmol) ofsodium cyanoborohydride at intervals of about 10 hours. After a total of64 hours at 37° C. 0.5 ml of HEPES pH 7.3 and 15 mg of dithiothreitol(DTT) were added. Fractionation by gel filtration (Sephadex G-10, 12×130mm Eluent: 20 mM NaCl) under argon yielded 3.6 ml of solution oflactosylated peptide in the free mercapto form (1.74 μmol correspondingto the Ellmann test; 84% yield). The samples of modified peptide showeda color reaction with anisaldehyde but no color reaction with nihydrin;this accords with the assumption that all 4 N-terminal amino groups arelactosylated. The tetra-galactose peptide-polylysine conjugate is shownin FIG. 6.

b) Preparation of 3-dithiopyridinepropionate-modified polylysine

400 μl of a 15 mM ethanol solution of SPDP (6.0 μmol) were added, withintensive mixing, to a gel-filtered solution of 0.60 μmol poly-L-lysinewith an average chain length of 290 lysine monomers (pL290,hydrobromide, Sigma) in 1.2 ml of 100 mM HEPES pH 7.9. 1 hour later, 500μl of 1 M sodium acetate pH 5 were added after gel filtration (SephadexG-25) with 100 mM sodium acetate, the solution contained 0.56 μmol pL290with 5.77 μmol of dithiopyridine linker.

c) Conjugation of the Peptide with Polylysine

Conjugates were prepared by mixing 1.5 μmol of the lactosylated peptideprepared in a) in 3 ml of 20 mM NaCl with 0.146 μl of the modified pL290obtained from b) in 620 μl of 100 mM sodium acetate buffer under anargon atmosphere. After the addition of 100 μl of 2 M HEPES pH 7.9, thereaction mixture was left to stand for 18 hours at ambient temperature.By the addition of NaCl, the salt concentration was adjusted to 0.66 Mand the conjugates were isolated by cation exchange chromatography(Pharmacia Mono S column HR 5/5; gradient elution, Buffer A: 50 mM HEPESpH 7.3; Buffer B: Buffer A plus 3 M NaCl). The product fractions elutedat salt concentrations of about 1.2 M-1.8 M and were pooled in twoconjugate fractions: the conjugate fractions were named gal4pL1 andgal4pL2. Dialysis against 25 mM HEPES pH 7.3 resulted in the conjugatefractions gal4pL1, containing 24 nmol of modified pL290 and gal4pL2,containing 24.5 nmol of modified pL290.

d) Preparation of asialofetuin conjugates

The conjugates were prepared on the same principle as the transferrinconjugates; a similar method of preparing asialoorosomucoid-polylysineconjugates was described by Wu and Wu in 1988; the disclosure of whichis fully incorporated by reference herein.

The coupling of asialofetuin to polylysine was carried out by bondingvia disulfide bridges after modification with the bifunctional reagentSPDP (Pharmacia). A solution of 100 mg (2.2 μmol) of asialofetuin(Sigma) in 2 ml of 100 mM HEPES pH 7.9 was subjected to gel filtrationon a Sephadex G-25 column. 330 μl of a 15 mM ethanolic solution of SPDP(5.0 μmol) were added to the resulting 4 ml solution with vigorousstirring. After 1 hour at ambient temperature, purification was carriedout by another gel filtration (Sephadex G-25); this resulted in 5 ml ofa solution of 1.4 μmol asialofetuin, modified with 2.5 μmol ofdithiopyridine linker.

Conjugates were prepared by mixing 1.4 μmol of asialofetuin in 5 ml of100 mM HEPES pH 7.9 with 0.33 μmol of modified pL190 (containing 1.07μmol of mercaptopropionate groups; the same process was used as for thepreparation of the transferrin conjugates) in 6.5 ml of 200 mM HEPES pH7.6, under an Argon atmosphere. The reaction mixture was left to standfor 24 hours at ambient temperature. The conjugates were isolated fromthe reaction mixture by cation exchange chromatography (Pharmacia MonoS-column HR 10/10; gradient elution, Buffer A: 50 mM HEPES pH 7.9;Buffer B: Buffer A plus 3 M sodium chloride) and sodium chloride wasadded until a final concentration of 0.6 M was achieved before loadingthe column. The product fraction eluted at a salt concentration of about1.5 M. Dialysis with HBS yielded conjugates containing 0.52 μmol ofasialofetuin, modified with 0.24 μmol of pL190.

e) Transfection of HepG2 cells with pRSVL-DNA complexes

HepG2 cells were grown in DMEM medium plus 10% FCS 100 I.U./mlpenicillin, 100 μg/ml streptomycin and 2 mM glutamine in T25 flasks.Transfections were carried out at a density of 400,000 cells per flask.Before the transfection, the cells were washed with 4 ml of fresh mediumcontaining 10% FCS. Immediately before the transfection, chloroquine(Sigma) was added so that the final concentration in the cell suspension(plus DNA solution) was 100 μM.

10 μg pRSVL-DNA in 330 μl HBS were, mixed with the quantities ofTfpL190B conjugate (TfpL), asialofetuin pL90 conjugate (AFpL),polylysine 290 (pL) or Tetra-galactosepeptide polylysine conjugategal4pL specified in FIG. 7 in 170 μl of HBS. In the competitionexperiments, 240 μg of asialofetuin ((gal)4pL+Af) or 30 μg lactosylatedpeptide ((gal)4pL+(gal)4) were added after 30 minutes. The mixture wasadded to the cells; the cells were incubated at 37° C. for 4 hours, thenthe transfection medium was replaced by 4 ml of fresh DMEM medium plus10% FCS. After 24 hours the cells were harvested for the luciferaseassay. The values given in FIG. 7, represent the total luciferaseactivity of the transfected cells as shown in the figure, pL and TfpLshow slight luciferase activities; (gal)4pL shows values as high asAfpL; (gal)4 or Af compete for the asialoglycoprotein receptor and, asexpected, lower the values.

f) Transfection of HepG2 cells with pCMVL-DNA complexes

HepG2 cells were grown in 6 cm plates to a cell density of 300,000 cellsper plate, as described in e). Before transfection, the cells werewashed with 1 ml of fresh medium containing 2% of FCS.

6 μg of pCMVL-DNA in HBS were mixed with the quantities of TfpL10Bconjugate (TfpL), asialofetuin-pL conjugate (AFpL), polylysine290(pLys290), (gal)4pL1 or (gal)4pl2 specified in FIG. 8, in 170 μl HBS.After 30 minutes, 1 ml of DMEM, containing 2% FCS and 50 μl adenovirusstock solution d1312C, were added to each DNA-conjugate complex. In thecompetition experiments, 30 μg of lactosylated peptide (gal)4pL((gal)4pL1+(gal)4 or (gal)4pL2+(gal)4) were added, as specified. Themixture was added to the cells; the cells were incubated for 2 hours at37° C., then 1.5 ml of medium, containing 10% FCS were added. Two hourslater, the transfection medium was replaced by 4 ml of fresh DMEM mediumplus 10% FCS. After 24 hours the cells were harvested for the luciferaseassay; the values in FIG. 8, represent the total luciferase activity ofthe transfected cells. pLys290 exhibits an effect, gal)4pL exhibits astronger effect; an addition of (gal)4, which competes for theasialoglycoprotein receptor, reduces the values to the value obtainedfor polylysine.

g) Transfection of TIBB73 cells with pCMVL-DNA complexes

Cells of the embryonic murine liver cell line ATCC TIB73 (BNL CL.2;Patek et al., 1978; the disclosure of which is fully incorporated byreference herein) were grown at 37° C. in a 5% CO₂ atmosphere in “highglucose” DMEM (0.4% glucose), supplemented with 10% heat-inactivated FCScontaining 100 I.U./ml penicillin, 100 μg/ml streptomycin and 2 mMglutamine in 6 cm plates.

The transfections were carried out at a cell density of 300,000 cellsper plate. Before the transfection, the cells were washed with 1 ml offresh medium plus 2% of FCS.

6 μg pCMVL-DNA in 300 μl HBS were mixed with the specified amounts ofmurine transferrin-polylysine290 conjugate (mTfpL), asialofetuin-pLconjugates (AFpL), polylysine290 (pLys290), (gal)4pL1 or (gal)4pL2 in170 μl HBS. After 30 minutes, 1 ml of DMEM, containing 2% FCS and 50 μlof adenovirus stock solution d1312 were added to each DNA conjugatecomplex. The mixture was added to the cells, the cells were incubatedfor 2 hours at 37° C., then 1.5 ml of medium containing 10% FCS. Twohours later, the transfection medium was replaced by 4 ml of freshmedium. After 24 hours the cells were harvested for the luciferaseassay; the values shown in FIG. 9B represent the total luciferaseactivity of the transfected cells.

As a comparison, transfection was carried out without adenovirus in thepresence of chloroquine: the transfection was performed at a celldensity of 300,000 cells per plate. Before the transfection, the cellswere washed with 1 ml of fresh medium containing 2% FCS. Immediatelybefore transfection, chloroquine (Sigma) was added so that the finalconcentration in the cell suspension (plus DNA-solution) was 100 μM. 6μg of pCMVL-DNA in 330 μl HBS were mixed with the specified amounts ofmTfpL, AFpL, pLys290, (gal)4pL1 or (gal)4pL2 in 170 μl of HBS. After 30minutes the DNA complexes were added to the cells. The cells wereincubated for 2 hours at 37° C., then 1.5 ml of medium containing 10% ofFCS and 100 μM chloroquine were added. Two hours later the transfectionmedium was replaced by 4 ml of fresh medium. After 24 hours the cellswere harvested for the measurement of luciferase. The values obtainedfor the luciferase activity are shown in FIG. 9A.

Example 7 Introduction of DNA in T Cells

a) Preparation of antiCD7 Polylysine190 conjugates

A solution of 1.3 mg of antiCD7 of antibody (Immunotech) in 50 mM HEPESpH 7.9 was mixed with 49 μl 1 mM ethanolic solution of SPDP (Pharmacia).After 1 hour at ambient temperature the mixture was filtered over aSephadex G-25 gel column (eluent 50 mM HEPES Buffer pH 7.9), therebyobtaining 1.19 mg (7.5 nmol) of antiCD7, modified with 33 nmolpyridyldithiopropionate groups. Poly(L)lysine190, fluorescent labelledusing FITC, was modified analogously with SPDP and brought into the formmodified with free mercapto groups by treating it with dithiothreitoland subsequent gel filtration. A solution of 11 nmol of polylysine190,modified with 35 nmol mercapto groups, in 0.2 ml of 30 mM sodium acetatebuffer was mixed with modified antiCD7 (in 0.5 ml 300 mM HEPES pH 7.9)with the exclusion of oxygen, and left to stand overnight at ambienttemperature. The reaction mixture was adjusted to a content of about 0.6M by the addition of 5 M NaCl. Isolation of the conjugates was carriedout by ion exchange chromatography (Mono S, Pharmacia, 50 mM HEPES pH7.3, salt gradient 0.6 M to 3 M NaCl); after dialysis against 10 mMHEPES pH 7.3, corresponding conjugates were obtained consisting of 0.51mg (3.2 nmol) of antiCD7-antibody, modified with 6.2 nmol polylysine190.

b) Preparation of gp120-Polylysine 190 conjugates

Coupling was carried out by methods known from the literature bythioether-linking after modification with N-hydroxysuccinimide ester of6-maleimidocaproic acid (EMCS, Sigma) (Fujiwara et al., 1981; thedisclosure of which is fully incorporated by reference herein).

Thioether-linked gp120-Polylysine 190-conjugates:

A solution of 2 mg of recombinant gp120 in 0.45 ml of 100 mM HEPES pH7.9 was mixed with 17 μl of a 10 mM solution of EMCS indimethylformamide. After 1 hour at ambient temperature, filtration wascarried out over a Sephadex G-25 gel column (eluent 100 mM HEPES-Buffer7.9). The product solution (1.2 ml) was immediately reacted, with theexclusion of oxygen, with a solution of 9.3 nmol Polylysine 190,fluorescence-labelled and modified with 30 nmol mercapto groups (in 90μl 30 mM sodium acetate pH 5.0), and left to stand overnight at ambienttemperature. The reaction mixture was adjusted to a content of about 0.6M by the addition of 5 M NaCl . The conjugates were isolated by ionexchange chromatography (Mono S, Pharmacia 50mM HEPES pH 7.3, saltgradient 0.6 M to 3 M NaCl); after fractionation and dialysis against 25mM HEPES pH 7.3, 3 conjugate fractions A, B and C were obtained,consisting of 0.40 mg of rgp120 modified with 1.9 nmol polylysine 190(in the case of Fraction A), or 0.25 mg rgp120 modified with 2.5 nmolpolylysine 190 (Fraction B), or 0.1 mg rgp120 modified with 1.6 nmol ofpolylysine 190 (Fraction C).

pCMVL-DNA (6 μg/sample) were complexed with the specified amounts ofpolylysine90 or the specified polylysine conjugates in 500 μl HBS. Inthe meantime, aliquots of H9 cells (10⁶ cells in 5 ml of RPMI with 2%FCS) or primary human lymphocytes (3×10⁶ cells in Iscove's modifiedDulbecco's medium (IMDM) plus 2% FCS) were prepared. The polylysine-DNAcomplexes were added to each cell sample. 5 minutes later, the specifiedamount of adenovirus d1312 was added. The cells were then incubated for1.5 hours at 37° C., then 15 ml of RPMI (in the case of H9 cells) orIMDM (in the case of the primary lymphocytes) plus 20% FCS were added toeach sample. The cells were incubated for 24 hours at 37° C., harvestedand treated as in the other examples, to determine the luciferaseactivity. The results of the tests carried out are given in FIG. 10A (H9cells) and FIG. 10B (primary lymphocytes): in H9 cells, the antiCD7conjugate (FIG. 10A, lanes 7 to 9) and the gp120 conjugate (lanes 10 to12) showed the best results in terms of the gene transfer achieved withadenovirus, while the gp120 conjugate achieved a clear expression of theluciferase gene even in the absence of adenovirus. It is worth notingthat, in the tests carried out, only the gp120 conjugate had the abilityto introduce DNA into primary lymphocytes, and then only in the presenceof defective adenovirus (FIG. 10B, lanes 7 and 8).

Example 8 Inactivation of Adenoviruses

a) UV Inactivation

An adenovirus d1312 preparation, prepared and stored as described in theintroduction to the Examples, was placed in 2 cm diameter wells of acell culture plate (300 μl per well) on ice at an 8 cm spacing from 2 UVlamps (Philips TUV15 (G15 T8) lamps). The virus was exposed to the UVradiation for the times specified in FIG. 11A and aliquots of eachpreparation were investigated for their virus titer and to determinewhether and to what extent they were capable of augmenting gene transferwith polylysine-transferrin conjugates into HeLa cells.

The cultivation of the cells and the transfection were carried outessentially as described above under “cells and media”; the componentsused for transfection are shown in FIG. 11A. The complexes of pCMVL-DNAand 12 μg TfpL were prepared in 500 μl HBS and added to 3×10⁵ HeLa cells(in 1 ml DMEM plus 2% FCS). About 5 minutes later, 54 μl of each viruspreparation was added to each culture and the culture was incubated at37° C. for one and a half to two hours. Then a 5 ml aliquot of DMEM plus10% FCS was added to each culture, incubation was continued at 37° C.for 24 hours and the cultures were harvested and investigated forluciferase activity. The quantity of 54 μl of non-irradiated virus isnot in the saturation range, i.e. the test is sensitive to a quantity ofvirus at least 3 times greater. The results obtained for the luciferaseexpression are shown in FIG. 11B (shaded rectangles). The virus titer ofeach preparation was determined using the E1a complementing cell line293. Serial dilutions of the non-irradiated and irradiated virus sampleswere prepared in DMEM plus 2% FCS. Parallel to this, samples of 5×10⁴293 cells were prepared (in a 2 cm well) in 200 μl DMEM plus 2% FCS. A5μl aliquot of each dilution was placed in each well. In order to allowthe virus to bind to the cells, incubation was carried out at 37° C. forone and a half hours, then 2 ml of DMEM plus 10% FCS were placed in eachwell. 48 Hours later, the cultures were examined in order to determinethe cytopathic effect. The virus dilution above which less than 50% ofthe cells in the culture show a significant cytopathic effect after 48hours indicates the relative amount of infectious virus in each viruspreparation. The results obtained are shown in FIG. 11B (openrectangles). The results of the tests carried out in this Example, showthe decrease of 4 logs in the virus titer resulting from UV radiation isassociated with only a twentyfold reduction in the luciferase genetransfer. This demonstrates that mechanisms which are crucial to theinfectivity of the virus can be destroyed without affecting the abilityof the virus to augment gene transfer.

It was observed that at low doses of the virus, the increase in genetransfer caused by the virus fell slightly (FIG. 11A, lanes 3 to 6) andthat this effect was more significant at the high doses (lanes 7 to 10).

b) Inactivation of Adenoviruses with Formaldehyde

2 ml of adenovirus preparation were passed over a 10 ml G25 column(Pharmacia Sephadex G 25, PD10), pre-equilibrated with 150 mM NaCl, 25mM HEPES pH 7.9, 10% glycerol, and taken up in a volume of 2.5 ml.Aliquots of the gel-filtered virus preparation were incubated without(0), with 0.01%, 0.1% or 1% formaldehyde for 20 hours over ice. ThenTris pH 7.4 was added to give a concentration of 100 mM, then thesamples were dialyzed first for 2 hours against 1 liter of 150 mM NaCl,50 mM Tris pH 7.4 and 50% glycerol and then overnight against 2×1 liter150 mM NaCl, 20 mM HEPES pH 7.9 and 50% glycerol.

Aliquots of the virus were then examined for their titer on 293 cells(CPE endpoint assay or plaque assay, Precious and Russel, 1985). Thenthe effect of the formaldehyde-treated viruses on gene transfer intoHeLa cells (300,000) was determined as in the previous examples bymeasuring the luciferase activity. 90 μl of the virus preparation,resulted in a DNA transfer corresponding to more than 10⁸ light units.Treatment of the virus with 0.01 % or with 0.1% formaldehyde resulted ina slight reduction in gene transfer activity (approximately tenfoldreduction at 0.1%). Although the treatment with 1% formaldehyde causes astriking loss of gene transfer activity, 90 μl of the virus was stillable to produce a gene expression corresponding to 10⁴ light units.

In the treatment with 0.1% formaldehyde, a reduction in the virus titerto 10⁵ PFU (plaque forming units) was coupled with a decrease in theluciferase activity of only 10%. The results of the test are shown inFIG. 12A.

c) Inactivation of Adenoviruses with long-wave UV+8-methoxy psoralentreatment

Aliquots of purified virus were adjusted to 0.33 μg/μl 8-methoxypsoralen (stock concentration 33 μg/μl 8-methoxy psoralen dissolved inDMSO) and exposed to a 365 nm UV light source (UVP model TL-33), on ice,at a distance of 4 cm from the lamp filter. Exposure to the UV light wasfor 15-30 minutes, as indicated in the figure legends. The virus sampleswere then passed over a Sephadex G-25 column (Pharmacia, PD-10)equilibrated with HBS+40% glycerol and stored at −70° C.

Viral preparations were tested for either their activity in augmentingpCMVL/hTfpL conjugate delivery into HeLa cells (as evidenced by theresulting light units of luciferase activity, right-hand axes FIG. 12B)or for the ability to replicate in 293 cells (viral titer, left-handaxes FIG. 12B).

In the Examples which follow, illustrating the increase in theinternalization of transferrin-polylysine-DNA complexes by means ofretroviruses, the following materials and methods were used:

Transferrin-polylysine190 conjugates and conjugate-DNA complexes wereprepared analogously to the preceding Examples.

NIH3T3 cells were grown in DMEM medium with the addition of 10% FCS, 100I.U./ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine. For thetransfections, 5 to 7×10⁵ cells per T25 were plated out 18 to 24 hoursbefore transfection. Immediately before transfection, the cells wereplaced in fresh medium and the various components used for transfectionwere added in the following order: Chloroquine (100 μM, where stated),polylysine-transferrin-DNA complex and retrovirus preparation. The cellswere then incubated for 4 hours at 37° C., and the medium was changedand the cells were harvested 24 hours later. Extracts were preparedusing three freeze/thaw cycles; aliquots of the extract, standardizedfor of protein content, were examined for luciferase activity as statedin the preceding Examples.

Example 9 Transfection of NIH3T3 Cells with Moloney Virus

Under the conditions specified, transfections of 10⁶ NIH3T3 cells werecarried out with TfpL-DNA complexes in the presence of 100 μMchloroquine or without chloroquine as shown in FIG. 13. It was foundthat without chloroquine the values for the luciferase activity reachedonly a background level (lane 1), whereas in the presence of chloroquinea high expression of the pRSVL reporter gene was measured (lane 2).Increasing amounts of the Moloney leukaemia virus (designated RVS in theFigure), which were added to the cells at the same time as the DNAcomplexes, were able to increase the luciferase gene expression stillfurther.

Example 10 Investigation into Whether the Gene Transfer Effect in theTransfection of NIH3T3 Cells with Transferrin-polylysine DNA ComplexesCan be Attributed to Moloney Virus

The virus preparation used in Example 9 was a crude, unfractionatedsupernatant of retrovirus expressing cells. In order to obtain evidencethat the increase in the DNA transfer achieved with this viruspreparation could actually be ascribed to the virus, the supernatant wassubjected to the dialysis/concentration purification described above,the retrovirus supernatant (shown as RVS in the drawing) beingconcentrated by a factor 10. If the retrovirus is responsible for theincrease, the activity retained by the membrane, apart from anyinactivation of the extremely unstable retrovirus during theconcentration step, should be approximately 10 times that of theoriginal supernatant. As in the previous Example, 10⁶ NIH3T3 cells weretransfected under the conditions given in FIG. 14. FIG. 14 shows thatthe gene transfer increasing effect is present in the membrane retentate(20 to 600 μl were used, lanes 3 to 6). It was also found that 200 and600 μl of the ten fold concentrated preparation are about half as activeas 2 or 6 ml of the original, unconcentrated retrovirus preparation(lanes 7 and 8). Parallel tests were carried out with human K562 cellshaving no receptor for the ecotropic murine retrovirus. As expected,there was no increase in gene expression.

Example 11 Interactions between Transferrin and its Receptor Play a Rolein the Gene Transfer Effect of Moloney Virus

In order to rule out the possibility that the transfer of TfpL/pRSVLcomplexes into the cells can be ascribed to non-specific binding ofpolylysine to the retrovirus, and in order to clarify the entrymechanism further, the retrovirus was examined for its ability totransport plasmid DNA, complexed only with polylysine, into the cell.The quantity of polylysine used corresponds to the optimum amountdetermined earlier which brings about total condensation of the plasmidDNA and is similar to the quantity of the polylysine used with thepolylysine-transferrin conjugate (Wagner et al., 1991a; the disclosureof which is fully incorporated by reference herein). The tests, theresults of which are shown in FIG. 15, demonstrated that the reportergene, in the absence of chloroquine, is not expressed either in the formof TfpL-RSVL complexes or in the form of pL-pRSVL complexes (lanes 1 and2). In the presence of the retrovirus, on the other hand, the reporterDNA applied as a TfpL complex was expressed, but not in the form ofpL-DNA complex (see lanes 3 and 4 together with lanes 5 and 6).Moreover, the tests carried out showed that the presence of excess freetransferrin resulted in the reduction in the DNA transfer facilitated bythe retrovirus (lanes 7 and 8). These results support the propositionthat interactions between transferrin and its receptor play an essentialpart in augmenting the DNA uptake effected by the retrovirus.

Example 12 Influence on pH on the Gene Transfer Effect of Retroviruses

The experiments carried out in this Example were performed in order toexamine the influence of the pH on the ability of retroviruses toaugment gene transfer. The transfection experiments were carried out asin the preceding Examples. The two well-characterized inhibitors ofendosome pH reduction, monensin and ammonium chloride, were used. Theexperimental results are shown in FIG. 16. The effect of the twosubstances on TfpL-DNA transfer was investigated and it was found thatneither of the two substances can functionally replace chloroquine.However, a slight increase in the luciferase gene expression was foundat higher ammonium chloride concentrations (lanes 1 to 5). Theretrovirus alone shows the slight augmentation in DNA transfer asobserved in the previous Examples (lane 6). A sharp increase wasobserved when the retrovirus was used in the presence of 1 μM monensin(lane 7). A less powerful effect was observed at a higher monensinconcentration (lane 8) and in the presence of ammonium chloride (lanes 9and 10).

Example 13 Augmentation of the Gene Transfer Achieved by TransferrinConjugates by Means of the N-terminal Endosomolytic Peptide of InfluenzaHemagglutinin HA2

a) Synthesis of the peptide

The peptide of the sequence (SEQ ID NO:1) of theGly-Leu-Phe-Glu-Ala-Ile-Ala-Gly-Phe-lle-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-Gly-Gly-Cyswas synthesized using the Fmoc (fluorenylmethoxycarbonyl) method(Atherton et al., 1979), using an Applied Biosystems 431A peptidesynthesizer. The side chain protecting groups were t-butyl for Cys, Gluand Asp and trityl for Asn. After the coupling reaction, a ninhydrintest was carried out which showed a coupling level of >98% for eachstep. Beginning with amino acid 19, double couplings were carried out.The N-terminal Fmoc group was removed from part of the peptide resinwith 20% piperidine in NMP (N-methylpyrrolidine). Then theFmoc-protected and unprotected fractions were washed with DCM(dichloromethane) and dried under high vacuum. The yields were 294 mgFmoc-free peptide resin and 367 mg of Fmoc-protected peptide resin. 111mg of the Fmoc-free peptide resin was subjected to trifluoroacetic acidcleaving for one and half hours using a mixture of 10 ml TFA, 0.75 g ofphenol, 300 μl of EDT (ethanedithiol), 250 μl of Et-S-Me(ethylmethylsulfide) and 500 μl of water. The peptide was filtered fromthe resin through a sintered glass filter. The resin was washed with DCMand added to the filtrate. The filtrate was concentrated down to about 2ml and then added dropwise with stirring to 40 ml of ether. The peptidedeposit was removed by centrifuging and the ether supernatant wasdiscarded. The precipitate was washed three times with 40 ml of etherand dried in a high vacuum. The 58 mg of crude product obtained weredissolved in 3.5 ml of 20 mM NH₄HCO₃, containing 300 μl of 25% NH₃/1.The solution was gel-filtered using the same buffer on a pre-packagedSephadex G-25 column (Pharmacia PD-10). All the material was placed on aMono Q column (Pharmacia 100×14 mm) gradient: 0-10 min 100% A, 10-100min 0-100% B. A: 20 mM NH₄HCO₃+300 μl NH₃/1. B: A+3 M NaCl. Measured at280 nm, Trp-fluorescence detection at 354 nm. Flow rate 1 ml/min. Theproduct is eluted with 1 M NaCl. The main fraction of the Mono Q column,was further purified by reverse phase HPLC using a BIORAD-Hi-Pore RP 304column (250×10 mm) (gradient 50 to 100% Buffer B in 12.5 min, 12.5 to 25min 100% B. A: 20 mM NH₄HCO₃+300 μl NH₃/1, B: A in 98% methanol. Flowrate: 3 ml/min. Measured at 237 nm). The product is eluted at 100% B.The product fractions were evaporated down in a Speedvac, re-dissolvedin buffer A and finally lyophilized. The yield was 8.4 mg of theHPLC-purified product in the cysteine-protected form. This peptide wasdesignated P16. In order to obtain P16 in the free mercapto form, thet-butyl-protected substance was treated for 30 minutes at ambienttemperature withthioanisol/edmanedithiol/trifluoraceticacid/trifluoro-methanesulfonicacid (2/1/40/3; trifluoromethanesulfonic acid was added in theproporation specified after the other components). The peptide wasisolated by ether precipitation and subsequent gel filtration (SephadexG-25) using the above mentioned buffer A under an argon atmosphere.

b) Coupling of the influenza peptide to polylysine

b1) Direct binding via SPDP (Succinimidylpyridyl-dithiopropionate)

19.8 mg of polylysine 300 hydrobromide (Sigma) were gel-filtered on aSephadex G-25 column (Pharmacia PD-10) in sodium acetate pH 5 in orderto eliminate the low molecular fractions. On the basis of the ninhydrintest, the pL concentration after gel filtration was 3.16 mg/ml. The pHof the solution was adjusted to 7-8 using 1 M NaOH. 0.64 μmol of SPDP(Pharmacia: 40 mM solution in absolute EtOH) were added to 2.5 ml of thepL solution (7.9 mg pL=0.13 μmol). This corresponds to a molar ratio ofSPDP:pL of 5:1. The mixture was left to react overnight and gel-filteredin 20 mM NH₄HCO₃ pH 8.2 on a G25 column. After reduction of 1 aliquot ofthe filtrate with DTT (dithiothreitol) the measurement of thiopyridoneshowed that the reaction was complete. 0.3 μmol of pL PDP-modified(based on μmol of PDP) in 2.2 ml were left to react with 0.35 μmol ofpeptide in the thiol form. A white precipitate which appeared when thepeptide and pL were mixed was dissolved by adjusting the solution to 2 Mguanidinium hydrochloride, the reaction taking place overnight.Photometric measurement of thiopyridone in the reaction mixture againconfirmed that the reaction was complete. The mixture was then dialyzedtwice against 2 liters of 20 mM HEPES/0.5 M guanidinium hydrochloride.The resulting solution was added to a Mono S column (0.7×6 cm,Pharmacia) (gradient: 0 to 20 min 100% A, 20-140 min 0-100% B. A: 20 mNHEPES pH 7.3/0.5 M guanidinium hydrochloride, B: 20 mM HEPES pH 7.3/3 Mguanidinium hydrochloride, 0.3 ml/min. Detection at 280 nm andfluorescence detection at 354 nm, excitation at 280 nm). The productfraction which was eluted with 1.5 M guanidinium hydrochloride wasdialyzed against 2 liters of HBS. Subsequent determination of the pLconcentration by the ninhydrin test showed a concentration of about 1.14mg/ml. The quantity of peptide in the solution of the conjugate wascalculated from its absorption at 280 nm; this gave a molar ratio ofpeptide:pL of 4:1.

b2) Binding via a polyethyleneglycol linker

14.6 mg of pL 300 hydrobromide (Sigma) were gel filtered as described inb1). According to the ninhydrin test, the pL concentration after gelfiltration was 4.93 mg/ml. The pH of the solution was adjusted to 7-8with 1 M NaOH. 4.33 μmol PDP (Pharmacia; 30 mM solution in absoluteEtOH) were added to 2.7 ml of pL solution (13.3 mg pL=0.22 μmol). Thiscorresponds to a molar ratio of PDP:pL of 20:1. After one and a halfhours the reaction mixture was gel filtered on a Sephadex G-25 column in0.1 M sodium acetate 3 M guanidinium hydrochloride. After reduction of 1aliquot of the filtrate with DTT, thiopyridone was determined,indicating that the product fraction contained 3.62 μmol of PDP. ThePDP-modified pL was reduced by adding 79 mg of DTT to the solution.After 2 hours reduction the solution was again filtered on G-25 underthe conditions specified. The thiol measurement using the Ellman testshowed a thiol concentration of 3.15 μmol in 2.224 ml.

17.6 mg=5 μmol POE (Polyoxyethylene-bis(6-aminohexyl), Sigma) weredissolved in 500 μl of 20 mM NaHCO₃/3 M guanidinium hydrochloride, pH7-8, and reacted with 13.8 mg of EMCS (ε-maleimidocaproicacid-N-hydroxysuccinimide ester) (Sigma) (=44.7 μmol), dissolved in 300μl DMF (dimethylformamide). After 30 minutes, the solution was gelfiltered on G-25 (20 mM NaHCO₃/3 M guanidinium hydrochloride).Photometric measurement of the maleimido group at 300 nm showed aconcentration of 6.36 μmol of reacted EMCS in 2 ml of solution.

1.39 μmol of the peptide in thiol form in (2.5 ml of 20 mM NaHCO₃/3 Mguanidinium hydrochloride) were added dropwise to 1.05 ml of thissolution (corresponding to 3.34 μmol EMCS) while the mixture wasintensively mixed with a vortex in an argon current. After 15 minutes nomore free thiol groups could be detected by the Ellman test.

The solution of the reduced mercapto-modified pL was adjusted to a pH of7-8 by the addition of 1 M NaOH. 1.37 ml of this solution were added tothe above reaction mixture while intensive mixing was carried out bymeans of a Vortex. This gave a molar ratio of peptide-SH:POE-EMCS:pL-SHof 1:2.4:1.4 (based on EMCS and SH). After 2.5 hours reaction, no morefree thiol groups could be detected by the Ellman test. The material wasdialyzed overnight against 2 liters of 20 mM HEPES pH 7.3/0.6 M NaCl andthen added to a Mono S column (gradient 0 to 20 min 22% A, 20-150 min22-100% B. A: 20 mM HEPES pH 7.3, B: A+3 M NaCl. Flow rate 0.3 ml/min.UV-measurement was carried out at 280 nm and fluorescence measurement at354 nm). The product which was eluted with 1.5 to 1.6 M NaCl wasdialyzed against 2 liters of HBS. The measurement of the pLconcentration using the ninhydrin test and photometric determination ofthe peptide concentration at 280 nm yielded a calculated pL ratio of12:1 at a pL concentration of 0.49 mg/ml in a total volume of 4.5 ml.

c) Liposome preparation

Using the REV method (reverse phase evaporation) liposomes were prepared(Szoka and Papahadjopoulos, 1978; Straubinger and Papahadjopoulos 1983;the disclosures of which are fully incorporated by reference herein):aqueous phase 10 mM HEPES pH 7.3; 100 mM calcein; 150 mM NaCl; organicphase: a solution of 300 μmol L-∝-lecithin (from egg yolk, chieflypalmitoyloleoylphosphatidylcholine; Avanti Polar Lipids) in 260 μl ofchloroform was evaporated down using a rotary evaporator. The materialwas then dried in a high vacuum and then dissolved again in 3 ml ofdiethylether. 1 ml of the aqueous phase was thoroughly washed with theether phase using a vortex and treated with ultrasound for 5 minutes at0° C. in a sonicator (bath type). After 30 minutes on ice, the materialwas treated with ultrasound for a further 10 minutes. The resultingstable emulsion was slowly evaporated down in a rotary evaporator. Afterthe diethylether had been eliminated at 100 mbar, 0.75 ml of the aqueousphase were added. Residual traces of diethylether were eliminated byfurther evaporation at 50 mbar for 30 minutes. The resulting preparation(1.7 ml) was centrifuged at 500 rpm. 1.0 ml thereof was extruded througha nucleopore polycarbonate membrane (0.1 μm), giving a final volume of0.7 ml liposome solution. The liposomes were separated from thenon-incorporated material by gel filtration (Sephadex G-50 medium,Pharmacia; 23 ml gel volume, 10 mM HEPES pH 7.3/150 mM NaCl). 6fractions of 500 μl were collected. Lipid phosphorus was determinedusing the method of Bartlett, 1959, at 2 mM.

d) Liposome Leakage Assay

The release of the liposome content (leakage) was measured by means ofthe emergence of the enclosed calcein and the resulting dilution whichstops the quenching of fluorescence (Bondeson et al., 1984). The calceinfluorescence was measured with a Kontron SMF 25 spectralfluorometer(excitation at 490 nm, emission at 515 nm). For this purpose, 100 μlaliquots of the above liposome solution were diluted 100 times with 0.1M sodium acetate/50 mM NaCl or 10 mM HEPES/150 mM NaCl buffer with thecorresponding pH (4.3, 4.5, 5.0, 6.0, 7.3) in order to obtain a value of1 ml. To these solutions were added 2.5 μl of the peptide(t-butyl-protected form; 1 μg/μl of solution in HBS) in cuvettes, whilemixing with a gentle stream of argon (final concentration 400 nMpeptide). The calcein fluorescence was measured at different times afterthe addition of the peptide. The values for 100% leakage were determinedby the addition of 2 μl Triton X-100 (Fluka).

The same procedure was used to measure the calcein fluorescence afterthe addition of peptide-pL conjugates to the liposome solution. 2.5 μgof the conjugate (1 μg/μl concentration based on the quantity of pLalone) were added to 1 ml of liposome solution (final concentration 20nM modified peptide). Similarly, 2.5 μg of peptide-polylysine conjugatewere subjected to the leakage assay after incubation with 5 μg DNA (15minutes).

It was found that the peptide only causes the release of the liposomecontent in the acidic range (FIG. 17). The peptide conjugate was activeat a substantially lower pH, while even at a neutral pH a strongactivity was found which was further increased as the pH was lowered.Complexing of the conjugate with DNA eliminated the activity at aneutral pH, whereas at an acidic pH there was a significant activity.

e) Transfection of K562-cells

K562-cells were grown in suspension in RPMI 1640 medium (Gibco BRL plus2 g sodium bicarbonate/l) plus 10% FCS, 100 I.U. per ml penicillin, 100μg/ml streptomycin and 2 mM glutamine up to a density of 500,000cells/ml. 12 to 20 hours before transfection the cells were placed infresh medium containing 50 μM desferrioxamine (this measure was taken toincrease the number of transferrin receptors). On the day oftransfection, the cells were collected, suspended in fresh mediumcontaining 10% FCS plus 50 μM desferrioxamine (250,000 cells per ml) and2 ml portions were placed in a dish with 24 wells.

6 μg of pCMVL-DNA in 160 μl HBS were mixed with the quantities of TfpLconjugate specified in FIG. 18 or with pL300 in 160 μl HBS, then after15 minutes the specified amounts of influenza peptide-pL-conjugate(P16pL) were added and after a further 15 minutes the mixture was addedto the K562 cells. The cells were incubated for 24 hours at 37° C. andthen harvested for the luciferase assay. The luciferase activity wasdetermined as specified in the previous Examples. The values given inFIG. 18 represent the total luciferase activity of the transfectedcells.

f) Transfection of HeLa cells

HeLa cells are cultivated in 6 cm culture dishes as described under“Cells and Media”. The transfections were carried out at a density of300,000 cells per plate. Before transfection, the cells were incubatedwith 1 ml of fresh medium containing 2% FCS. 6 μg of pCMVL-DNA in 160 μlHBS were mixed with the quantities of TfpL conjugate specified in FIG.19 or with pL300 or a mixture of both in 160 μl HBS. After 15 minutes,the specified amounts of influenza peptide-pL-conjugates (P16pL) wereadded and after a further 15 minutes the mixture was added to the cells.The cells were incubated for 2 hours at 37° C., then 2.5 ml of freshmedium were added with an additional 10% FCS. The cells were incubatedfor 24 hours at 37° C. and then harvested for the luciferase assay. Theluciferase activity was determined as described in the precedingExamples. The values given in the figure represent the total luciferaseactivity of the transfected cells.

Example 14 Augmentation of the Gene Transfer Achieved by TransferrinConjugates by Means of a Second N-terminal Endosomolytic Peptide ofInfluenza Hemagglutinin HA2

a) Synthesis of influenza peptide-polylysine conjugate

The peptide of the sequence (SEQ ID NO:2)Gly-Leu-Phe-Gly-Ala-Ile-Ala-Gly-Phe-Ile-Glu-Asn-Gly-Trp-Glu-Gly-Met-Ile-Asp-Gly-Gly-Gly-Cys(designated P41) was synthesized in the same way as the peptidedescribed in Example 13, a). The coupling of the influenza peptide topolylysine (pL300) was performed as in Example 13, b1) by binding viaSPDP. Thereby conjugates (P41pL) with a molar ratio of peptide:pL of 4:1were obtained.

b) Transfection of HeLa cells

HeLa cells were grown in DMEM medium plus 5% FCS, 100 units/mlpenicillin, 100 μg/ml streptomycin and 2 mM glutamine in 6 cm plates.Transfections were performed at a density of 300,000 cells per plate.Before the transfection, cells are incubated with 1.5 ml of fresh mediumcontaining 2% FCS.

6 μg pCMVL-DNA in 160 μl HBS (150 mM NaCl, 20 mM HEPES 7.3) were mixedwith 6 μg of TfpL190B conjugate in 160 μl HBS, after 15 min 10 μginfluenza peptide-pL-conjugate P41pL or, for comparison, 18 μg ofinfluenza peptide-pL-conjugate P16pL (see Example 13) were added (FIG.20); the specified amounts of the two peptide conjugates had been testedto be optimal amounts for the augmentation of the gene transfer. Afterfurther 15 min the mixture was added to the cells. The cells wereincubated at 37° C. for 4 hours, then 2 ml of medium containing 18% FCSwere added. After 24 hours the cells were harvested for the luciferaseassay. Values as shown in FIG. 20 represent the total luciferaseactivity of the transfected cells.

The comparison of the experiments with the two peptide conjugates showsa more than 3.5 fold higher augmentation of the gene transfer obtainedwith the second peptide conjugate P41pL.

c) Transfection of BNL CL.2 cells with influenza peptide polylysineconjugates

BNL CL.2 cells were grown as described in Example 6. Influenza peptideP41 was conjugated with polylysine 300 at a molar ratio of peptide topolylysine of 1:1, 3:1 and 8:1. Complexes of 6 μg pCMVL-DNA and 20 μg ofthe conjugates were added to the cells. For comparison, 20 μg of pL₃₀₀or 20 μg of P16 polylysine conjugate, prepared as described in Example13, were used. The cells were incubated at 37° C. for 4 h, then 2 ml ofmedium containing 18% FCS was added. After 24 h, the cells wereharvested for the luciferase assay, the results of which are shown inFIG. 20B. The content of peptide in the conjugates correlated with theaugmentation of gene expression. In the liposome leakage assay (FIG.20C), which was performed as described in Example 13, the activity ofthe conjugates (at pH 5, equivalents to 2.5 μg polylysine) increasedwith their content of peptide. (In the figure, P41 is designated“influ2”)

Example 15 Transfection of HeLa Cells With a β-galactosidase ReporterGene Construct and In Situ Demonstration of β-galactosidase Expression

a) Culturing and transfection of cells

For the transfection, HeLa cells were grown in DMEM medium containing 5%FCS, penicillin, streptomycin and glutamine, as described in theprevious Examples, in 3 cm culture dishes on cover slips (3×10⁴ cellsper dish).

For the transfection, 6 μg of the β-galactosidase reporter geneconstruct (pCMV-β-gal) in 160 μl of HBS were complexed with 12 μg ofTfpL190B in 160 μl of HBS and incubated for 30 minutes at ambienttemperature.

In another experiment, 6 μg of pCMV-β-gal in 160 μl of HBS wereincubated with 6 μg of TfpL190B in 80 μl of HBS for 15 minutes atambient temperature. Then 12 μg of the influenza peptide conjugate(P16pL) prepared in Example 13 in 80 μl of HBS were added and themixture was incubated for a further 15 minutes. These DNA-polycationcomplexes were then mixed with 1 ml of DMEM plus 2% FCS, antibiotics andglutamine, as described above. In order to demonstrate the effect ofchloroquine and adenovirus on the success of the transfection, inadditional experiments chloroquine was also added to the mediumcontaining the DNA polycation complexes, in a final concentration of 100μM or 50 μl of the adenovirus strain solution d1312.

For the transfections, the original culture medium was removed from thecells and 1 ml of medium containing the DNA complexes with or withoutchloroquine or virus was added. After an incubation period of 2 hours at37° C., 1 ml of DMEM containing 10% FCS, antibiotics and glutamine wasadded to the cells and incubation was continued for a further 2 hours.Then all the medium was removed and the cells were cultivated in 3 ml offresh DMEM plus 10% FCS, antibiotics and glutamine.

b) β-galactosidase assay

48 hours after transfection, the medium was removed, the cells werewashed once with phosphate-buffered saline solution (PBS) and fixed with0.5% glutardialdehyde in PBS for 5 minutes at ambient temperature. Thenthe fixative was discharged and the cells were washed once with PBS.Then incubation was carried out with the staining solution (10 mMphosphate buffer pH 7.0, 150 mM NaCl, 1 mM MgCl₂, 3.3 mM K₄Fe(CN)₆3H₂O,3.3 mM K₃Fe(CN)₆ and 0.2% 5-bromo4-chloro-3-indolyl-β-galactopyranoside)at 37° C. for 20 minutes to 3 hours (Lim and Chae, 1989). Then the coverslips were rinsed in PBS, water and 96% ethanol, dried and mounted inMowiol on slides. A Zeiss Axiophot Microscope was used for analysis.

FIG. 21 shows images of the microscopic magnifications (112 times). A:HeLa cells transfected with 6 μg pCMV-β-gal, complexed with 12 μgTfpL190B. The staining reaction for β-galactosidase was carried out for3 hours. The Figure shows that very few cells (55 cells; a group ofstained cells is indicated by an arrow) express the β-galactosidasegene. B: HeLa cells transfected with 6 μg pCMV-β-gal, complexed with 6μg TfpL190B and 12 μg P16pL. Staining reaction: 3 hours. Few cells (250cells) express the β-galactosidase gene. However, die reaction of thecells is stronger than in A. C: HeLa cells transfected with 6 μgpCMV-β-gal, complexed with 6 μg TfpL190B and 12 μg P16pL in the presenceof 100 μM of chloroquine. Staining reaction: 3 hours. Numerous groups ofcells show a strongly positive reaction (more than 1,000 cells). D: HeLacells transfected with 6 μg pCMV-β-gal, complexed with 12 μg TfpL190B inthe presence of adenovirus d1312. Staining 20 reaction 20 min. Nearlyall the cells (more than 90%) show a positive reaction. E:Non-transfected HeLa cells (control for the specificity of theβ-galactosidase reaction). Staining reaction: 3 hours.

Example 16 Transfection of HeLa Cells With a 48 kb Cosmid in thePresence of Adenovirus

a) Preparation of a cosmid containing the luciferase coding sequence

A 3.0 kb SalI fragment, containing a single P. pyralis luciferase codingsequence under control of the RSV promoter, was isolated from theplasmid p220RSVLucα and ligated into the unique SalI site of the cosmidclone C1-7aA1 to form concatamers. (C1-7aA1 comprises a 37 kb humangenomic DNA Sau3A fragment (partial digest), encoding no apparent genes,cloned into the BamHI site of the cosmid vector pWE15 (Stratagene)). Theligation reaction product was then packaged in vitro and an aliquot ofthe resulting phage particles infected into E. coli NM544 and platedonto LB amp plates. The recombinants were screened by colonyhybridization, using the 3.0 kb SalI fragment (³²P labelled by randompriming) as a hybridization probe, and a number of positives analyzed byrestriction mapping. A cosmid construct (CosLuc) containing a singlecopy of the SalI insert was grown and purified on a CsCl gradient (totalsize=48 kb).

A small control cosmid pWELuc (12 kb) was prepared by digesting CosLucwith NotI, religating, transforming bacteria and isolating a clonecontaining the appropriate plasmid. This resulted in a 12 kb DNAmolecule lacking the human DNA insert and part of the polylinker ofCosLuc. The plasmid pSPNeoLuc (8 kb) is the plasmid described in Example5 which contains an RSV-luciferase gene fragment (an Apa1/Pvu1 fragmentof pRSVL, cloned into the Cla1 site of the pUCμ Locus).

b) Delivery of the cosmid into HeLa cells

HeLa cells (3×10⁴ cells per 6 cm dish) covered with 1 ml DMEM+2% FCSwere incubated with TfpL/DNA complexes prepared as described in theMaterials and Methods section, containing the indicated quantities ofhTfpL, free pL and DNA. Cell incubation mixtures included, in addition,either 100 μM chloroquine (lanes 1 and 2) or 10 μl adenovirus d1312containing 5×10¹¹ particles per ml, (lanes 3-12). After a 2 hourincubation at 37° C., 4 ml of DMEM+10% FCS was added to each dish; 24hours later, cells were harvested and luciferase activity was measured.Results are shown in FIG. 22A.

c) Delivery of the cosmid into Neuroblastoma cells

Cells of a neuroblastoma cell line designated GI-ME-N (Donti et al.,1988) (1×10⁶ cells per 6 cm dish) covered with 1 ml DMEM+2% FCS wereincubated with TfpL-DNA complexes prepared as described in the Materialsand Methods section, containing the indicated quantities of hTfpL, freepL and DNA. Cell incubation mixtures included, in addition, either 100μM chloroquine (lanes 3 and 4) or 10 μl adenovirus d1312 containing5×10¹¹ particles per ml, (lanes 5 and 6). After a 2 hour incubation at37° C., 4 ml of DMEM+10% FCS was added to each dish; 24 hours later,cells were harvested and luciferase activity was measured. Results areshown in FIG. 22B.

Example 17 Gene Transfer by Means of Chemically CoupledAdenovirus-polylysine Conjugates

a) Preparation of adenovirus-polylysine conjugates by chemical coupling

2.35 ml of a gel filtered (Sephadex G-25 PD10, Pharmacia) solution ofadenovirus d1312 (approx. 10¹¹ particles) in 150 mM NaCl/25 mM HEPES, pH7.9/10% glycerol was mixed with 10 μl (10 nmol) of a 1 mM solution ofSPDP (Pharmacia). After 3.5 hours at ambient temperature the modifiedvirus was separated from the excess reagent by gel filtration (asabove). The solution (2.5 ml) was purged with argon and allowed toreact, under the exclusion of oxygen, under argon, with 42 μl of asolution of FITC-labelled polylysine (1 nmol), modified with 2.3 nmol ofmercaptopropionate groups (prepared as described in EP 388 758). After18 hours at ambient temperature half the solution was transferred into acentrifuge test-tube, carefully covered with 1 ml of a cesium chloridesolution (density 1.33 g/ml) and centrifuged at ambient temperature for2 hours at 35000 rpm (SW60 rotor). The virus band was collected as 200μl cesium chloride fraction and diluted to 1 ml with HBS/50% glycerol. ADNA binding assay was carried out with 300 μl of the modified virus: thevirus solution was diluted with 1 ml HBS and mixed with 100 μl ofsolution of a ³⁵S-labelled DNA (15 ng pRSVL, prepared by Nicktranslation). As a control, the experiment was carried out in parallelwith the same amount of unmodified virus d1312. After 30 minutes thesamples were transferred into centrifuge tubes, carefully covered with 1ml of a cesium chloride solution (density 1.33 g/ml) and centrifuged for2 hours at 35000 rpm (SW60 rotor) at ambient temperature. The gradientwas divided into 5 fractions; fraction 1, 1 ml; fraction 2, 0.6 ml,fractions 3-5, 200 μl each. The radioactivity of 200 μl portions of thefractions was measured and is shown in FIG. 23. The fractions whichcontain virus (3-5), especially fraction 3, show a significantly higherradioactivity than the control. This can be attributed to specificassociation of the polylysine-modified adenovirus with the labelled DNA,the presence of cesium chloride possibly causing partial dissociation ofthe complexes.

b) Transfection of K562 cells

K562-cells (ATCC CCL 243) were grown in suspension in RPMI 1640 medium(Gibco BRL, plus 2 g sodium bicarbonate/l 10% FCS, 100 units per mlpenicillin, 100 μl/μl streptomycin and 2 mM glutamine) up to a densityof 500,000 cells/ml. 12 to 20 hours before transfection the cells wereplaced in fresh medium containing 50 μM desferrioxamine (this measurewas taken to increase the number of transferrin receptors). On the dayof transfection, the cells were collected, suspended in fresh mediumcontaining 10% FCS plus 50 μM desferrioxamine (250,000 cells per ml) and2 ml portions were placed in a dish with 24 wells.

The specified amounts of pCMVL-DNA (6, 0.6, 0.06 μg) in 100 μl of HBSwere mixed with 50 μl of polylysine adenovirus (pLadeno) orcorresponding amounts (35 μl) of control adenovirus d1312. After 20minutes, corresponding amounts (12, 1.2, 0.12 μg) of TfpL190B conjugatein 150 μl of HBS were added. After a further 20 minutes the mixture wasadded to the K562 cells. The cells were incubated for 24 hours at 37° C.and then harvested for the luciferase assay. The luciferase activity wasdetermined as in the preceding Examples. The values given in FIG. 24represent the total luciferase activity of the transfected cells.

c) Transfection of HeLa cells

One method of testing the activity of a polylysine-virus conjugate is bychecking the conjugate for its ability to transport very small amountsof DNA (less than 0.1 μg). An increased DNA transfer capacity wasexpected when the adenovirus is directly bound to thepolylysine-condensed DNA, as the internalizing factors (transferrin andadenovirus (protein)) are directly associated with the DNA which is tobe transported. To test this assumption, a constant quantity of thepolylysine-adenovirus conjugate (2.5 μl, about 5×10⁷ virus particles)was completed with different amounts (3 μg to 0.0003 μg) of reporterplasmid in 475 μl of HBS. After 15 minutes incubation at ambienttemperature a quantity of transferrin-polylysine corresponding to themass of DNA was added to each sample (this quantity of TfpL was selectedbecause it guarantees total packaging (electroneutrality) of 50% of theplasmid DNA and at the same time ensures binding space for thevirus-polylysine conjugate. After the addition of TfpL the mixtures wereincubated for 15 minutes, then each mixture was placed in a 6 cm culturedish containing 300,000 HeLa cells in 1 ml of DMEM/2% FCS. Then thecells were incubated for 1.5 hours at 37° C., then 4 ml of DMEM/10% FCSwere added. In parallel, equivalent quantities of DNA were completedwith a two-fold mass excess of TfpL (the quantity for total DNAcondensation) and used for the gene transfer into HeLa cells (once onits own and once in the presence of 25 μl of the non-polylysine-coupledadenovirus d1312 preparation). After 24 hours the cells were harvested,extracts were prepared and aliquots were examined for luciferaseactivity. The results of these tests are shown in FIG. 25: in theabsence of adenovirus, no luciferase activity can be detected in aquantity of DNA less than 0.3 μg. Both polylysine coupled andnon-coupled adenovirus functioned well with large quantities of DNA (3μg and 0.3 μg). However, with the non-coupled adenovirus there was anapproximately 100 fold fall in activity at 0.03 μg and negligibleactivity below this amount of DNA. By contrast the polylysine-coupledvirus retains its gene transfer capacity both at 0.003 and at 0.0003 μgof DNA. This quantity of DNA corresponds to about 150 DNA molecules percell and about 1 virus particle per cell.

Example 18 Gene Transfer by Means of Adenoviruses Enzymatically Coupledto Polylysine

a) Enzyme reaction

2 ml of the adenovirus preparation (stain d1312; 5×10¹⁰ PFU/ml) wereapplied to a Sephadex G-25 gel filtration column (Pharmacia)equilibrated with 25 ml of reaction buffer (0.1 M Tris-HCl; pH 8.0, 2 mMDTT, 30% glycerol). Elution was carried out with 3.5 ml of reactionbuffer. The reaction mixture for enzymatic coupling consists of 1150 μlof the virus elution fraction, 0.5 nmol guinea-pig livertransglutaminase (TG) (Sigma), 2 nmol or 20 nmol of Polylysine290, 10 mMCaCl₂ and reaction buffer in a final volume of 1500 μl. The reaction wascarried out at 37° C. for 1 hour and then stopped by the addition of 30μl of 0.5 M EDTA. In order to monitor the specificity of the coupling,reaction mixtures were also prepared without transglutaminase.Non-incorporated polylysine was separated from the viruses bycentrifuging in a CsCl-gradient (density 1.33 g/ml; 170,000x g, 2hours). The fraction containing the viruses was collected, mixed with anequal volume of glycerol, frozen in liquid nitrogen and stored at −70°C.

b) Demonstrating the binding of polylysine to adenoviruses

The reaction was carried out as described above with polylysine whichhad been labelled with ¹²⁵I with Bolton-Hunter reagent (Amersham). Afterthe CsCl-gradient centrifugation the virus fraction was drawn off andseparated by means of another CsCl gradient. The gradient was thenfractionated and the radioactivity in every fraction was determinedusing a scintillation counter. As shown in FIG. 26, it became apparentthat in the reaction mixture with TG (d1312/TG-pL), radioactivepolylysine had accumulated in the virus fraction (virus). In the controlmixture without TG (d1312/pL) there was no accumulation of radioactivepolylysine in the virus fraction.

c) Testing the polylysine-modified adenovirus fractions for their effecton the efficiency of transfection

i) Cells and media

For the transfection, 5×10⁵ cells (murine hepatocytes; ATCC No.: TIB 73)in DMEM with 10% heat-inactivated fetal calf serum (FCS), 2 mMglutamine, 100 I.U./ml penicillin and 100 μg/ml of streptomycin wereseeded in 6 cm culture dishes.

ii) Formation of the virus-DNA-transferrin complexes

50 μl of the polylysine-modified virus fraction were mixed with 6 μg ofthe DNA plasmid pCMVL in 10 μl HBS and incubated for 20 minutes atambient temperature. Then 8 μg of murine transferrin-polylysine290B(mTfpL) were added to the mixture and incubation was continued for afurther 20 minutes.

iii) Transfection of the murine hepatocytes

The virus-DNA-transferrin complexes were mixed with 1.5 ml of medium(DMEM with 2% FCS, 2 mM glutamine and antibiotics) and added to thecells, after removal of the old medium. After 2 hours incubation at 37°C., 2 ml of DMEM with 10% FCS, glutamine and antibiotics were added tothe cells. After a further 2 hours cultivation the entire medium wasremoved and 4 ml of fresh DMEM with 10% FCS, glutamine and antibioticswere added to the cells.

iv) Determining the luciferase expression

24 hours after transfection the cells were harvested and the luciferaseassay was carried out as described above. As can be seen from FIG. 27,the virus preparation in which the adenoviruses had been treated with TGand 20 nmol of polylysine (d1312/TG-20 nmol pL) showed the strongestexpression (153540000 light units). The virus preparation with TG and 2nmol of polylysine (d1312/TG-2 nmol pL) was somewhat less active(57880000 light units). The control fraction in which the adenoviruseswere treated with 20 nmol of polylysine but with no TG was lesseffective by a factor of 500 approximately. As a comparison, furthercomplexes were used for transfection with the initial preparation ofadenoviruses treated neither with TG nor with polylysine (d1312). Thispreparation yielded 4403000 light units.

(d) Increasing the transfection efficiency by polylysine-modifiedadenoviruses compared with unmodified adenoviruses, particularly withsmall amounts of DNA

Transfection was carried out as described in Example c), using 50 μl ofthe adenovirus fraction d1312/TG-20 nmol pL and 6 μg pCMV-Luc/8 μgmTfpL, 0.6 μg pCMVL(=pCMV-Luc)/0.8 μg mTfpL or 0.06 μg pCMV-Luc/0.08 μgmTfpL for complexing. As a comparison, transfections were also carriedout with 6 μg, 0.6 μg, 0.06 μg pCMV-Luc/mTfpL complexes and unmodifiedadenoviruses (d1312). It was found that the complexes withpolylysine-modified adenoviruses yielded high expression levels evenwith small amounts of DNA, whereas expression was sharply reduced withunmodified adenoviruses (FIG. 28).

Example 19 Gene Transfer With Conjugates in Which the Binding Betweenthe Adenovirus and Polylysine is Obtained by Means of aBiotin-streptavidin Bridge

a) Biotinylation of adenovirus d1312

2.4 ml of a gel filtered (Sephadex G-25 PD10, Pharmacia) solution ofadenovirus d1312 (about 10¹¹ particles) in 150 mM NaCl/5 mM HEPES, pH7.9/10% glycerol, was mixed with 10 μl (10 nmol) of a 1 mM solution ofNHS-LC biotin (Pierce 21335). After 3 hours at ambient temperature thebiotin-modified virus was separated from the excess reagent by gelfiltration (as above). The solution was adjusted to a glycerolconcentration of 40% by adding glycerol (total volume 3.2 ml) and storedat −25° C. The biotinylation of the virus was demonstrated byqualitative detection after dropwise addition of various dilutions ontocellulose nitrate membrane: after drying at 80° C. for 2 hours in avacuum dryer, blocking with BSA, incubating with streptavidin-conjugatedalkaline phosphatase (BRL), washing and incubating for 1 hour with thedeveloping solution NBT/X-phosphate(nitroblue-tetrazoliumsalt/5-bromo-4chloro-3-indolylphosphate, toluidine salt; BoehringerMannheim) a positive color reaction was found.

b) Preparation of streptavidin-polylysine conjugates

The coupling of streptavidin to polylysine was effected using the methoddescribed by Wagner et al., 1990, and in EP-A1 388 758. 79 nmol (4.7 mg)of streptavidin in 1 ml of 200 mM HEPES pH 7.9 and 300 mM NaCl weretreated with a 15 mM ethanolic solution of SPDP (236 nmol). After 1.5hours at ambient temperature the modified protein was gel filtered overa Sephadex G-25 column, thereby obtaining 75 nmol of streptavidin,modified with 196 nmol of dithiopyridine linker. The modified proteinwas reacted under an argon atmosphere with 3-mercaptopropionate-modifiedpolylysine (75 nmol, average chain length 290 lysine monomers, modifiedwith 190 nmol mercaptopropionate linker) in 2.6 ml of 100 mM HEPES pH7.9, 150 mM NaCl. Conjugates were isolated by cation exchangechromatography on a Mono S HR5 column (Pharmacia). (Gradient: 20-100%buffer B. Buffer A: 50 mM HEPES pH 7.9; buffer B: buffer A plus 3 Msodium chloride). The product faction eluted at a salt concentration ofbetween 1.2 M and 1.7 M. Dialysis against HBS (20 mM HEPES pH 7.3, 150mM NaCl) resulted in a conjugate consisting of 45 nmol of streptavidinand 53 nmol of polylysine.

c) Transfection of HeLa cells

HeLa cells were grown in 6 cm culture dishes as described in Example 13.The transfections were carried out at a density of 300,000 cells perplate. Before the transfection the cells were incubated with 1 ml offresh medium containing 2% FCS.

6 μg of pCMVL-DNA in 100 μl HBS were mixed with 0.8 μg ofstreptavidin-polylysine in 170 μl of HBS. After 20 minutes, 3 μg ofpolylysine pL300 in 170 μl of HBS were added. After another 20 minutes,65 μl of biotinylated adenovirus or, as the control, correspondingamounts of adenovirus d1312 (30 μl, starting virus for modification),were added. The complex mixtures, (“biotinAdV/complex A” or “controlAdV” see FIG. 29) were left to stand for a further 20 minutes.

Alternative complexing was carried out by mixing 65 μl of biotinylatedadenovirus first with 0.8 μg of streptavidin-polylysine in 50 μl HBSthen adding 6 μg of pCMVL-DNA in 170 μl of HBS after 20 minutes, and afurther 20 minutes later adding 3 μg of polylysine pL300 in 200 μl HBS.(Complex mixture “biotinAdV/complex B”).

0.6 μg of pCMVL-DNA in 67 μl HBS were mixed with 0.3 μg ofstreptavidin-polylysine in 33 μl of HBS. After 20 minutes, 65 μl ofbiotinylated adenovirus or, as the control, corresponding quantities ofadenovirus d1312 (30 μl, starting virus for modification) were added.The complex mixtures (“biotinAdV/complex A” or “control AdV”, see FIG.29) were left to stand for a further 20 minutes and then diluted to 500μl with HBS. Alternative complexing was carried out by mixing 65 μl ofbiotinylated adenovirus first with 0.3 μg of streptavidin-polylysine in50 μl of HBS and after 20 minutes adding 0.6 μg of pCMVL-DNA in 50 μlHBS. The complex mixture (“biotinAdV/complex B”) was left to stand for afurther 20 minutes and then diluted with HBS to 500 μl.

The mixtures were added to the cells, the cells were incubated for 2hours at 37° C., then 2.5 ml of fresh medium containing 10% added FCSwere added. The cells were incubated for 24 hours at 37° C. and thenharvested for the luciferase assay. The luciferase activity wasdetermined as described in the preceding Examples. The values given inFIG. 29 represent the total luciferase activity of the transfectedcells.

In parallel, transfections of HeLa cells were carried out using as thevirus component of the conjugate a biotinylated virus which had beeninactivated by psoralen/UV-treatment Inactivation was carried out asfollows: 200 μl batches of biotinylated virus preparation were placed intwo wells of a 1.6 cm tissue culture plate. 2 μl (33 mg/ml) of8-methoxypsoralen (in DMSO) were added to each sample, the dish wasplaced on ice and irradiated for 10 minutes with a UV lamp (365 nm; UVPTL-33 lamp) with the sample being 4 cm from the filter. After theirradiation the two samples were combined and gel filtered (G50, Nickcolumn, Pharmacia), the column having previously been equilibrated with40% glycerol in HBS. Aliquots of 75 μl were complexed with 0.8 μg ofstreptavidin-polylysine and used for the transfection of HeLa cells asdescribed above.

By cytopathic end point assay it was established that the virus titerwas reduced by a factor of more than 10⁴ by the inactivation, whereasthe transfer capacity was reduced by less than 50% at highconcentrations and by a factor of 5 at low concentrations.

d) Transfection of K562 cells

K562 cells were grown in suspension in RPMI 1640 medium (Gibco BRL, plus2 g sodium bicarbonate per 1 liter) plus 10% FCS, 100 units/mlpenicillin, 100 μg/ml streptomycin and 2 mM glutamine reaching a densityof 500,000 cells/ml. At 16 hours before transfection, the cells wereplaced in fresh medium containing 50 μM desferrioxamine (Sigma). Themorning of the transfection, the cells were collected, resuspended infresh medium containing 10% fetal calf serum (plus 50 μMdesferrioxamine) at 250,000 cells per ml, and placed in a 24-well dish,2 ml per well.

Three different types of DNA complexes were prepared: a) A solution of 6μg pCMVL-DNA in 160 μl HBS (150 mM NaCl, 20 mM HEPES 7.3) was mixed with12 μg of TfpL190B conjugate in 160 μl HBS, after 30 min 20 μl ofadenovirus d1312 was added and the mixture was added to K562 cells. b) Asolution of 800 ng streptavidin-polylysine in 160 μl HBS was mixed with20 μl of biotinylated adenovirus, prepared as described in a), after 30min a solution of 6 μg pCMVL-DNA in 160 μl HBS was added, and afterfurther 30 min the solution was mixed with 10 μg TfpL190B conjugate in160 μl HBS. After 30 min the mixture was added to the cells. c) The DNAcomplexes were prepared analogously to b) with the difference thatinstead of TfpL190B conjugate a solution of 3.5 μg poly(L)lysinep(Lys)290 was added.

The cells were incubated at 37° C. for 24 hours and then harvested forthe luciferase assay. Values as shown in FIG. 30 represent the totalluciferase activity of the transfected cells.

Example 20 Gene Transfer Into Primary Bone Marrow Cells

a) Isolation of Primary Bone Marrow Cells

Primary bone marrow cells were harvested from mice by flushing culturemedium (IMDM containing 10% FCS, 5×10⁻⁵ M β-mercaptoethanol, 1% IL-3conditioned medium and antibiotics) with an injection needle (0.4 mm or0.5 mm in diameter) attached to a 1 ml syringe through the isolatedfemura and tibiae. The cells were then washed once in culture medium bycentrifugation at 100 xg for 8 min. Thereafter the cells wereresuspended at a concentration of 10⁷ cells/ml and seeded into T25culture flasks. After 4 h the non-attached cells were transferred into anew T25 culture flask and cultured overnight in the presence of 50 μMdesferrioxamine.

b) Formation of adenovirus-DNA-transferrin complexes

For formation of adenovirus-DNA-transferrin complexes 50 μl ofbiotinylated adenovirus were incubated with 400 ng ofstreptavidin-modified polylysine in 20 μl HBS for 20 min. Then 20 μl ofHBS containing 6 μg pCMVL were added. After an incubation period for 20min 7 μg of mouse transferrin-polylysine conjugate (mTfpL) in 160 μl HBSwere added and the whole mixture was incubated for further 20 min.

c) Transfection

For transfection the bone marrow cells were recovered from the culturemedium of the T25 flask by centrifugation at 100 xg for 8 min. The cellpellet was resuspended in 3 ml of culture medium containing 2% FCS and250 μl of the adenovirus-DNA-transferrin complexes, and cultured in anew T25 flask for 3 h at 37° C. Then 3 ml and after a period of 2 hfurther 6 ml of culture medium containing 10% FCS were added.

d) Determination of luciferase expression

The bone marrow cells were harvested 48 h after transfection andanalyzed for expression of luciferase as described above. Thetransfection led to an expression of luciferase activity correspondingto 310×10³ light units/100 μg total cell protein.

Example 21 Transfection of Neuroblastoma Cells With a 48 kb Cosmid inPresence of Adenovirus

a) Preparation of a cosmid containing the luciferase coding sequence

A 3.0 kb Sal I fragment, containing a single P. pyralis luciferasecoding sequence under the control of the RSV promoter (De Wet et al.,1987; the disclosure of which is fully incorporated by referenceherein), was ligated in to the unique Sal I site of the cosmid cloneC1-7aA1 to form concatamers. (C1-7aA1 comprises a 37 kb human genomicDNA Sau 3A fragment (partial digest), encoding no apparent genes, clonedinto the Bam HI site of the cosmid vector pWE15 Stratagene)). Theligation reaction product was then packaged in vitro and an aliquot ofthe resulting phage particles infected into E. coli NM544 and plated onto LB amp plates. The recombinants were screened by colonyhybridization, using the 3.0 kb Sal I fragment (³²P labelled by randompriming) as a hybridization probe, and a number of positives analyzed byrestriction mapping. A cosmid construct (CosLuc) containing a singlecopy of the Sal I insert was grown and purified on a CsCl gradient(total size=48 kb).

A small control cosmid pWELuc (12 kb) was prepared by digesting CosLucwith Not I, religating, transforming bacteria and isolating a clonecontaining the appropriate plasmid. This resulted in a 12 kb DNAmolecule lacking the human DNA insert and part of the polylinker ofCosLuc.

b) Delivery of the cosmid into Neuroblastoma cells

Cells of a Neuroblastoma cell line designated GI-ME-N (Donti et al.,1988) (1×10⁶ cells per 6 cm dish) covered with 1 ml DMEM+2% FCS wereincubated with TfpL/DNA complexes prepared as described in materials andmethods section, containing the indicated quantities of hTfpL, free pLand DNA. As indicated, cell incubation mixtures included, in addition,either 100 μM chloroquine Canes 3 and 4) or 10 μl adenovirus d1312containing 5×10¹¹ particles per ml, (lanes 5 and 6). The last twosamples (indicated as StpL/Biotin) contained 15 μl biotinylatedadenovirus d1312 (1×10¹¹ particles) incubated withstreptavidin-polylysine (0.8 μg prepared as in Example 19) for 30minutes in 150 μl HBS. 6 μg DNA in 150 μl HBS was then added to thesample for 30 minutes, room temperature, followed by 150 μl HBScontaining 6 μg hTfpL+1 μg free pL. After a further 30 minutes roomtemperature incubation the mixture was added to the cells. After a 2hour incubation at 37° C., 4 ml of DMEM +10% FCS was added to each dish;24 hours later cells were harvested and luciferase activity wasmeasured. Results are shown in FIG. 31.

Example 22 Gene Transfer to Primary Airway Epithelial Cells EmployingMolecular Conjugate Vectors

Initial studies evaluating the feasibility of the use of gene transferemploying the molecular conjugate vectors of the invention for geneticcorrection of cystic fibrosis demonstrated that immortalized cell linesderived from human airway epithelium exhibited susceptibility to thisgene transfer method. To exclude the possibility that this phenomenonwas the result of immortalization-induced alterations of the airwayepithelium, transferrin-polylysine molecular conjugates were alsoevaluated in human primary respiratory epithelium cells (1° AE).

1° AE cells were obtained from nasal polyp specimens of patients asdescribed by Yankaskas, J. R. et at., 1987; the disclosure of which isfully incorporated by reference herein. Briefly, the tissues are rinsedin sterile saline, then in Joklik's Minimum Essential Medium (MEM) plusantibiotics (penicillin 50 U/ml, streptomycin 50 μg/ml, gentamicin 40μg/ml) and transported to the laboratory at 4° C. Cartilage and excesssubmucosal tissue are dissected free, and the epithelial sheets areincubated in protease solution (Sigma, type 14, 0.1 mg/dl) in MEM at 4°C. for 16 to 48 hours (Wu, R, 1985). Fetal bovine serum (FBS, 10%) isadded to neutralize the protease, and cells are detached by gentleagitation. The resulting suspension is filtered through 10 μm nylon meshto remove debris, pelleted (150 x g, 5 min) and washed in F12+10% FBS.

The 1° AE cells were then treated with transferrin-polylysine conjugates(hTfpL) containing a luciferase encoding plasmid (pRSVL) as a reportergene (L.U.). In this analysis, the primary cells did not exhibit thesusceptibility to this vector exhibited by the correspondingimmortalized cell lines (1° AE: background =429±41;+hTfpL=543±L.U.),likely indicating a relative paucity of transferrin receptors on 1° AE.

To exploit an alternative target receptor on 1° AE, conjugates wereconstructed that incorporated a replication-incompetent adenovirus asthe ligand moiety (bAdpL; see Example 19 a) and 19 b) for thepreparation of biotinylated adenovirus and streptavidin-polylysineconjugates; the luciferase encoding plasmid was used as the reportergene). Human 1° AE treated with this conjugate exhibited levels ofreporter gene expression significantly greater than background (+bAdpL=2585753±453585 L.U.). In addition, 1° AE derived from other speciesalso exhibited a high level of susceptibility to gene transfer by thisroute (mouse=3230244±343153; monkey=53498880±869481 L.U.). Thus, theability to accomplish gene transfer to 1° AE establishes the potentialutility of this approach to achieve the direct in vivo gene delivery.

Example 23 Gene Transfer to Hepatocytes with Molecular Conjugate Vectors

a) Transfection of tissue culture cells

Cells of the murine embryonic hepatocyte cell line BNL CL.2 (ATCC TIB73) were grown as described in Example 6. HeLa cells and hepatocyteswere grown in 6 cm plastic petri dishes. Transfection was carried out ata cell density of approximately 3×10⁵ cells per dish. Prior totransfection, 1 ml of fresh medium containing 2% FCS replaced thestandard culture medium.

b) Formation of binary complexes

Biotinylated adenoviruses (approx. 10⁹ PFUs; see Example 19 a) and 19b)) were reacted with 800 ng streptavidinylated polylysine in 50 μl HBS.After 30 min at room temperature, 6 μg pCMVL-DNA in 170 μl HBS wereadded, incubated for 30 min and then 3 μg polylysine pL300 in 200 μl HBSwas added and after further 30 min the solution used for transfectionexperiments.

c) Formation of Ternary complexes

Ternary DNA complexes containing adenovirus and transferrin were formedas follows: biotinylated adenoviruses (approx. 10⁹ viral particles) weremixed with 800 ng streptavidinylated polylysine. After 30 min at roomtemperature the solution was mixed with 6 μg plasmid DNA in 170 ml HBS,incubated for 30 min, then 10 μg transferrin-polylysine TfpL 190B(Wagner, E. et al., 1991b) in 200 μl HBS was added and after further 30min the solution was used for transfection experiments.

d) β-galactosidase assay

BNL CL.2 cells were seeded on to cover slips and 24 h later the cellswere transfected with the pCMV-β gal (Lim, K. and Chae, 1989) reportergene. 48 h later, β-galactosidase was assayed according to Lim and Chae.

Binary transport complexes. Biotinylated adenovirus was combined withstreptavidinylated polylysine. Alternatively adenovirus was linkedcovalently with polylysine through the action of transglutaminase.Adenovirus-polylysine conjugate was added to DNA allowing complexformation between DNA and polylysine, thus neutralizing a known fraction(ca. ¼) of the negative charges of the DNA. A calculated amount ofpolylysine was then added to neutralize the remainder of the charges.The complexes consisting of DNA bound to adenovirus-polylysine conjugateand to polylysine are referred to as binary transport complexes.

There are essentially two ways of assembling binary transfer complexes.DNA can be bound to streptavidinylated polylysine and then coupled tobiotinylated adenovirus or the adenovirus is coupled to polylysine firstand later complexed with DNA. The latter procedure quite clearly yieldsbetter results, especially at low DNA input, and therefore is thepreferred method for assembling both binary and ternary complexes.

Ternary transport complexes containing transferrin. Theadenovirus-polylysine conjugates were added to DNA allowing complexformation and neutralization of a fraction (approx. ¼) of the negativecharges of the DNA. A calculated amount of transferrin-polylysineconjugates was then added to complex and neutralize the remainder of theDNA. Complexes consisting of DNA, adenovirus-polylysine andtransferrin-polylysine are referred to as ternary complexes. Inprinciple, such a ternary complex should have the capacity of beingendocytosed by binding to either the cellular adenovirus receptors or totransferrin receptors.

Linkage between DNA condensates and adenovirus greatly enhancesluciferase reporter gene expression. Physical linkage between adenovirusstrain d1312 and polylysine can be brought about by either incubatingthe two components with transglutaminase or by biotinylation of theadenovirus and streptavidinylation on the polylysine. The effect oflinkage on transfection efficiency is clearly demonstrated in FIG. 32where hepatocytes were incubated with transferrin-polylysine DNAcomplexes (TfpL) in the presence of chloroquine or in the presence ofadenovirus (AdenoV+TfpL). Transferrinfection (this term designatestransferrin-mediated transfection) in the presence of free adenovirus iselevated showing the typical enhancement of release oftransferrin-polylysine DNA complexes into the cells. In slotpLAdenoV/TpfL adenovirus was conjugated with polylysine by means oftransglutaminase and was then reacted with DNA neutralizing part of thenegative charges of the DNA. Later, transferrin-polylysine was addedneutralizing the remainder of the charges. In this way, a ternarycomplex of adenovirus-polylysine/transferrin-polylysine/DNA wassynthesized. As can be seen, an extraordinary high value of 1.5×10⁹luciferase light units was obtained (or approx. 5000 light units percell). In slot adenoV+pL+TfpL, adenovirus and pL were mixed as for thetransglutaminase treatment. However, to demonstrate the specificity ofthe transglutaminase mediated binding of polylysine to the virus, theenzyme was omitted. Then the virus preparation was completed to the sameamount of DNA and TfpL as in pLAdenoV/TfpL. In this case, thetransfection was moderate as in AdenoV+TfpL because in both experimentsco-localization of virus and transferrin DNA is a stochastic process, incontrast to slot pLAdenoV/TfpL where co-internalization is assured bythe physical linkage of virus and DNA in a ternary complex yield highlevel of transferrinfection.

Transfection of K562 cells reveals the endosomolytic properties ofadenovirus. The human erythroleukemic cell line K562 contains ca.150,000 transferrin receptors (Klausner, R. D. et al., 1983b). In thepresence of chloroquine, as reported earlier (Cotten, M. et al., 1990),these cells can be transferrinfected at very high level withpolylysine-transferrin reporter DNA complexes even in the absence ofadenovirus (TfpL FIG. 33). The same complexes with added freeadenovirus, but in the absence of chloroquine, yield relatively poorlevels of reporter gene expression (AdenoV/TfpL) presumably because K562cells like other blood cells (Silver, L. et al., 1988; Horvath, J. etal., 1988) have low levels of adenovirus receptors. When the adenovirusis linked to polylysine via biotin/streptavidin bridge and the reporterDNA fully condensed by addition of more polylysine to complete thebinary complex (pLAdenoV/pL), adenovirus supported transfection reachesintermediate levels. Presumably, the few adenovirus receptors on K562cells are used efficaciously. If however the coupledadenovirus-polylysine-reporter DNA is fully condensed and neutralized byaddition of polylysine-transferrin to form a ternary complexpLAdenoV/TfpL and the numerous cellular transferrin receptors come intoplay, the transfection efficiency, owing to both efficient transferrinbinding and to the endosomolytic properties of the virus, is increasedby at least another 2 orders of magnitudes (FIG. 33).

Terry DNA complexes lead to the expression of the reporter gene inalmost 100% of hepatocytes. The efficacy of the novel DNA transportcomplexes were also tested in mouse hepatocytes (BNL CL.2), determiningthe percentages of the cells which can be reached with our varioustransfection protocols. A β-galactosidase gene driven by a CMV promotorwas used as a reporter gene. After fixation of the cells .-galactosidaseactivity was deed according to Lim and Chae, 1989.

FIG. 34 shows the β-galactosidase assay on mouse hepatocytes after a)transferrinfection in the presence of chloroquine; b) transferrinfectionin the presence of free d1312 adenovirus and c) transfection withternary, linked (d1312) adenovirus-polylysine-transferrin-reporter DNAcomplexes. In the absence of adenovirus, after standardtransferrinfection of the reporter DNA, only few cells express thereporter gene. The percentage of transfection is less than 0.1%. Whenchloroquine is included the percentage is increased to about 0.2% (FIG.34a). With free adenovirus about 5-10% of the cells express the reportergene (FIG. 34b) while the ternary complexes with transglutaminasemodified virus lead to expression in most, if not all, cells (FIG. 34c).Because the ternary complexes can be used at high dilution, the toxiceffect seen with high doses of free (inactivated) adenovirus does notusually arise. But it should be noted that where ternary complexes aredeployed at high concentration in order to reach 100% of the tissueculture cells, a similar toxic effect becomes noticeable. The toxiceffects may be caused by residual viral gene activity, the endosomolyticproperties of the added virus or is simply a consequence of the veryhigh level of expression of the transfected gene.

Expression of a transfected reporter gene is transient but lasts forweeks in non-dividing hepatocytes. Ternary sport complexes(pLAdenoV/TfpL) were made with d1312 polylysine adenovirus and d1312modified adenovirus further inactivated by reacting the virus withpsoralen. A 2/3 confluent hepatocyte cell culture was transfected as inFIG. 34b with the luciferase reporter gene CMVL and luciferase activitywas determined at different time points. As can be seen from FIG. 35,luciferase activity was maximal after 3 days at which time thehepatocyte cell culture became confluent and the cells stopped dividing.Expression of the reporter gene persisted in the non-dividing cellculture without applying selection for the maintenance of the gene andlasted for at least 6 weeks, especially when psoralen inactivatedadenovirus was used for the formation of the ternary transportcomplexes.

Example 24

The Use of the Chicken Adenovirus CELO to Augment DNA Delivery to HumanCells

In this example, the chicken adenovirus CELO was tested for its abilityto augment DNA delivery into human HeLa cells in a fashion analogous tothe above experiments employing the human adenovirus 5 experiments.

The chicken adenovirus CELO (Phelps strain, serotype FAV-1,7, chickenkidney cell passage) was used in these experiments. The virus (2 ml) waspassed through a PD-10 gel filtration column equilibrated with 20 mMHEPES pH 7.3, 150 mM NaCl, (HBS)+10% glycerol and 2 ml of the eluent wasreacted with 20 μl 1 mM NHS-LC-biotin (Pierce) for 3 hours at roomtemperature. The biotinylated virus was subsequently dialyzed against3×300 ml of HBS +40% glycerol at 4° C. and subsequently stored inaliquots at −70° C.

HeLa cells (5×10⁵ cells per 6 cm dish) were incubated in 2 ml of DMEM+2%FCS with 6 μg of the plasmid pCMVL complexed with polylysine (pLys) ortransferrin-polylysine (TfpL) mixtures in 500 μl HBS (the complexes werepre-incubated for 30 minutes at room temperature). The samples were thenadded to the cells at 37° C. in the presence of the quantity of virusindicated in FIG. 36. With 33 the samples containing biotinylated CELOvirus, the indicated quantity of virus was preincubated with theindicated quantity of streptavidin-polylysine (StrL) in 200 μl HBS for30 minutes at room temperature before adding 6 μg of the plasmid pCLucin 100 μl HBS. After a 30 minute zoom temperature incubation, theindicated quantity of TfpL material was added to the cells at 37° C. Twohours later, 5 ml of DMEM+10% FCS was added to the cells and 24 hourslater the cells were harvested and processed for luciferase assay. Theresults are shown in FIG. 36.

As shown in FIG. 36, the CELO virus as a free entity augmented DNAdelivery into HeLa cells (lanes 1-6. However, when the CELO virus wasmodified with biotin and included in a complex with streptavidin, eitherwith or without additional transferrin-polylysine the virus was found toaugment DNA delivery at a level that is comparable to when the humanadenovirus d1312 is employed. The particular line of HeLa cells displaysa high binding capacity for polylysine/DNA complexes in the absence oftransferrin (compare the luciferase activity of samples 1 and 4 in FIG.36), thus, inclusion of the CELO virus in a polylysine DNA complex issufficient to trigger uptake of the virus.

Example 25 Transfection of Myoblasts

a) Transfection of myoblasts and myotubes withDNA/transferrin-polylysine complexes in the presence of free adenovirusand in the presence of biotin/streptavidin-coupled adenovirus

C2C12 myoblasts Blau et al., 1985; ATCC No.: CRL 1772) and G8 myoblasts(ATCC No.: CRL 1456) were grown in high glucose DMEM plus 10% FCS.Myoblast cultures were transfected at subconfluence with ca. 5×10⁵ cellsper 6 cm dish. Myotube cultures were prepared by plating myoblasts in 6cm dishes (ca. 5×10⁵ cells per dish) and changing the medium to highglucose DMEM plus 2% horse serum when the cells reach confluence (Barrand Leiden, 1991; Dhawan et al., 1991). Myotube transfections wereperformed 5-7 days later. The transfection complexes were prepared asdescribed in Example 19 using the indicated quantities of TfpL, StrpLand biotinylated adenovirus d1312. The cells were harvested 20 hourspost-transfection and processed for luciferase activity measurement. TheFIG. 37 indicates the resulting luciferase activity, in light units, forthe entire cell sample: both myoblast and myotube cultures could betransfected with high efficiency. Upon differentiation to myotubes therewas less than one log decrease in transfection efficiencies (C2C12) orno significant decrease (G8). The participation in myotube formationoccurred at a lower frequency with the G8 line which may partly accountfor the lack of a detectable decrease in efficiencies in thedifferentiated culture. The role of the transferrin/transferrin receptorinteraction in the DNA delivery to this type of cell was not major. Inall four cell preparations there was only weak delivery of DNA usingTfpL/DNA complexes in the presence of free adenovirus d1312 (lanes1,4,7,10). Transfection efficiencies were enhanced using the coupledvirus system (lanes 2,3,5,6,8,9,11,12). There was a less than 1 logincrease in efficiencies comparing the delivery with combinationcomplexes containing only virus and polylysine/StrpL condensed tocomplexes which include transferrin-polylysine (compare, for example,lanes 2, no transferrin, with lane 3, transferrin). The poortransfection with free virus and the high transfection with coupledvirus complexes either in the presence of absence of transferrin-pLsuggest that the adenovirus serves as the ligand in these cells and inthe absence of coupling, the free virus may enter cells but the TfpL/DNAcomplex does not enter productively. (Me DNA used in this Example waspCMVL, designated pCluc in the Figure.)

b) Histochemical analysis of transfection frequencies in myotubes

C2C12 myotube cultures (5×10⁵ cells, as myoblasts, seeded per 6 cm dishand differentiated into myotubes) were prepared as in a). With the freevirus samples, pCMVβ-gal DNA (6kg) was complexed with 8 μg TfpL in 500μl HBS and supplied to the cells in the presence of 18 μl of adenovirusd1312 (1×10¹² virus per ml) in 2 ml of DMEM/2% FCS. Coupled virussamples were prepared with pCMVLacZ DNA (6 kg) complexed with 7 μg TfpLand 800 ng of StrpL plus 18 μl of biotinylated adenovirus d1312 (1×10¹²virus per ml) in 500 μl HBS and supplied to cells in 2 ml of DMEM/2%FCS. After a 24 hour incubation cells were stained forbeta-galactosidase activity, as described in Example 15.

The beta-galactosidase staining patterns were consistent with theresults of transfections using luciferase as the reporter gene (see a).Very low gene expression was obtained in myotube cultures using the freevirus while coupling the virus and DNA result in high level geneexpression. The presence of blue-stained, multi-nucleated tubulesindicated the successful transfer of a gene to these differentiatedcells in the presence of free adenovirus.

c) Delivery of DNA to mouse primary myoblast and myotube cultures

The major skeletal muscles from the both hind legs of a 4 week-old maleC57B1/6 mouse were sterilely isolated into PBS and minced intoapproximately 5 mm pieces. The tissue was suspended in 20 ml of PBS,allowed to settle for ca. 2 minutes and the supernatant was aspirated.This washing was repeated 3 times. The tissue was then mixed with 3.5 mlof PBS plus 0.5 ml of trypsin/EDTA, 0.5 ml of 1% (w/v) collagenase (typeII, Sigma),and 0.5 ml of 1% BSA (fraction V, in 4 mM CaCl₂) and allowedto incubate at 37° C. for 30 minutes with frequent, gentle agitation Atthe end of the 30 minute incubation the remaining tissue was allowed tosettle and the supernatant was removed and mixed with 5 ml of DMEM+20%FCS. The incubation with protease was repeated 3-4 times until thetissue was completely dispersed. The cell suspension was then passedthrough a cell strainer (Falcon) to remove any aggregates and tissuefragments, and centrifuged at 500 g for 15 minutes. The cell pellet wasresuspended in 10 ml of DMEM+20% FCS and the fibroblasts were removed byplating the cells on a 15 cm diameter, uncoated tissue culture dish for60 minutes. The unattached cells were then carefully removed and platedon 5 laminin-coated, 10 cm tissue culture dishes with 15 ml of DMEM+20%FCS per dish. Upon reaching confluence (approximately one week later)the cells were trypsinized and replated on laminin-coated, 6 cm dishes,approximately 1×10⁶ cells per dish. To generate myotube cultures,approximately 5 days later (when the cells had reached confluence) themedium was changed to DMEM+2% horse serum and one week latertransfections were performed. Myoblast cultures for transfection weretransfected in 6 cm dishes at approximately 80% confluence.

Laminin-coated cell culture plates were prepared in the followingmanner. Cell culture dishes were coated with 0.025 mg/ml polylysine (MW30,000-70,000, Sigma) in sterile water for 30 minutes at roomtemperature. The plates were rinsed 3 times with sterile water and airdried. The plates were then coated with 8 μg/ml laminin (EHS, Sigma) inwater overnight at room temperature. Plates were then washed 3 timeswith sterile water before seeding cells.

The DNA complexes used for transfections were prepared by diluting theindicated quantity of psoralen/UV-inactivated biotinylated adenovirusd1312 (prepared as described in Example 19) in 150 μl of HBS and adding1 μg of StrpL in 150 μl of HBS followed by a 30 minute, room temperatureincubation. HBS (100 μl) containing 6 μg of pCMVL (designated pCluc inthe Figure)was then added to each sample followed by another 30 minuteroom temperature incubation. Finally, 7 μg of TfpL in 100 μl of HBS wasadded to each sample, incubated for 30 minutes at room temperature andthen supplied to either myoblast or myotube cultures in 6 cm dishescontaining 2 ml of DMEM+2% FCS. After a 1 hour incubation, the mediumwas replaced with 5 μl of DMEM+20% FCS (myoblasts) or of DMEM+2% horseserum (myotubes) and the cells were harvested for luciferase analysis 48hours later. The luciferase activity from the entire cell sample isdisplayed in FIG. 38.

Example 26 Improvement of CELO Virus Delivery to Myoblasts Using aLectin Ligand

a) Comparative analysis of Adenovirus d1312 and CELO virus in HeLa andC2C12 myoblasts

Samples of either HeLa cells or C2C12 myoblasts (5×10⁵ cells per 6 cmdish) were transfected with 6 μg pCMVL (designated pCluc in the Figure)complexed with 1 μg StrpL/7μg TfpL plus 5 μl of biotinylated Adenovirusd1312 (see Example 19, 1×10¹² particles/ml) or 18 μl of biotinylatedCELO virus (see Example 24, 0.3×10¹² particles per ml). After a 20 hourincubation the cells were harvested and processed for luciferaseactivity measurement. FIG. 39 indicates the resulting luciferaseactivity from each entire cell sample.

Transfection into HeLa cells could be performed with comparableefficiencies using either the human adenovirus d1312/StrpL/TfpL/DNAcomplexes, which can enter the cells by either the adenovirus receptoror the transferrin receptor or the CELO virus/StrpL/TfpL/DNA complexeswhich can enter via the transferrin receptor. However, while delivery ofDNA into C2C12 myoblasts could be performed efficiently with adenovirusd1312 complexes, complexes containing the CELO virus functioned poorlyin these cells. Previous examples have demonstrated that the transferrinreceptor plays only a minor role in combination complex delivery tothese cells; presumably the adenovirus receptor is the major site ofentry. The poor activity of the CELO virus in myoblast might then be dueto a poor binding of both the CELO virus and transferrin to the C2C12myoblasts.

b) Improvement of CELO virus C2C12 myoblast transfection using wheatgerm agglutinin as a ligand

Due to the poor delivery obtained in a), a new ligand was selected toreplace transferrin.

Biotinylated wheat germ agglutinin (24 moles biotin per mole of protein)was purchased from Boehringer Mannheim. Biotinylated CELO virus wasprepared as previously described. Complexes containing 6 μg pCMVL plusthe indicated quantities of StrpL, TfpL, biotinylated wheat germagglutinin (WGA-B) and CELO virus were prepared in the following manner.Virus and WGA were diluted, together, in 150 μl HBS. StrpL was alsodiluted in 150 μl HBS and the two solutions were mixed and incubated atroom temperature for 30 minutes. The DNA, diluted in 100 μl of HBS, wasadded to the StrpL/Virus/WGA solution followed by another 30 minute roomtemperature incubation. Finally, TfpL in 100 μl HBS was added to themixture and again the sample was incubated at room temperature for 30minutes. The complexes were supplied to C2C12 myoblasts (5×10⁵ cells per6 cm dish) in 2 ml of DMEM plus 2%FCS. One hour later 5 ml of DMEM plus10% FCS was added to the cells and 20 hours later the cells wereprocessed for luciferase activity measurement. The activity (lightunits) in each entire cell sample is displayed in FIG. 40. (The DNA usedin this Example was pCMVL, designated pCluc in the Figure.)

Very poor DNA delivery was obtained in the absence of virus either withor without the WGA (lanes 1,6). Moderate delivery was obtained withcoupled CELO virus (lane 2) however a 16-fold increase in delivery wasobtained if WGA-B is included in the complex. Increasing the quantity ofWGA in the complex (from 1 μg to 5 μg) resulted in a slight decrease indelivery (compare lanes 3 and 4) while increasing the StrpL content ofthe complex (from 1 μg to 2 μg) enhanced the delivery slightly (comparelanes 3 and 5). These results clearly indicate that WGA-B as a ligandenhances CELO virus-mediated DNA delivery to C2C12 cells.

d) Expression of a full length factor VIII gene in C2C12 myoblast andmyotube cultures

C2C12 myoblast and myotubes cultures were prepared as described above.Transfections were performed using 6 μg of a plasmid encoding thefull-length human factor VIII cDNA (Wood et al., 1984; Eaton et al.,1986) complexed with 5 or 15 μl of biotinylated adenovirus (as indieplus 0.5 or 1 μg StrpL, and 7 or 6 μg of TfpL in the standard complexformation protocol.

The DNA/virus complexes were supplied to cells in 2% FCS/DMEM. After a 4hour incubation at 37° C., 3 ml of fresh DMEM+10% FCS was added to eachdish. 18 hours later the medium was harvested and assayed for thepresence of factor VIII using a COATEST, (KABI, Pharmacia) test systemwith an international standard as a reference. Factor VIII levels areplotted as mUnits generated per 24 hours, per 1×10⁶ cells (FIG. 41).

Example 27 Use of Adenovirus Protein for DNA Delivery

Adenovirus wt300 was grown in HeLa cells, purified and biotinylated asdescribed for adenovirus d1312. 1.2 ml of virus was dialyzed against 3×300 ml of 5 mM MES, 1 mM EDTA pH 6.25, 4° C., for 18 hours. Thematerial was then centrifuged for 30 minutes at 27 K in an SW60 rotor.The supernatant was carefully removed, the pellet was resuspended inHBS/40% glycerol. HEPES, pH 7.4 and NaCl were added to the supernatantto 20 mM and 150 mM, and both the pellet (containing the viral core andthe bulk of the hexon capsid, “core” in FIG. 42) and the supernatantfractions (containing the vertices, “vertices” in FIG. 42) were testedfor DNA delivery activity into both Mov13 mouse fibroblasts (Strauss andJaenisch, 1992) or HeLa cells.

Complex formation with DNA was performed in the following manner. Theindicated quantities of each fraction, disrupted virus beforecentrifugation or intact, virus (expressed as μg protein as determinedby a Bradford assay) were diluted in 300 μl HBS. Streptavidin-polylysine(3 μg in 50 μl HBS) was then added followed by a 30 minute roomtemperature incubation. 6 μg pCMVL (designated pCluc in the Figure) wasdiluted in 100 μl HBS and added to the first solution for a 30 minuteincubation. Finally, 2 μg of TfpL in 100μl of HBS was added followed byanother 30 minute incubation. In samples prepared with only TfpL, 8 μgof TfpL in 170 μl HBS was mixed with 6 μg of pCMVL in 330 μl of HBS for30 minutes at room temperature. The indicated quantities of virusprotein were diluted into 300 μl of HBS and then added to the TfpL/DNAcomplexes. All samples were then added to 5×10⁵ cells in 6 cm dish,containing 2 ml of DMEM/10%FCS (either HeLa of Mov13 fibroblasts) for 1hour. 5 ml of fresh medium containing 10% FCS was then added and thecells were processed for luciferase activity 20 hours later. Theresulting luciferase activity (in light units) is displayed in FIG. 42for both HeLa cells (panel A) or Mov13 fibroblasts panel B).

With both cell types there is a dose-dependent increase in DNA deliveryactivity associated with the vertex fraction (sample 4-6 in bothpanels). When the same quantity of biotinylated virus protein isincluded with TfpL/DNA complexes lacking streptavidin-polylysine DNAdelivery close to background levels is observed (sample 3 in eachpanel).

Example 28 Enhanced Gene Transfer Using DNA Ternary Complexes ContainingGalactose-ligand Conjugate

a) Ternary complexes containing influenza peptide conjugate

The presence of polylysine-conjugated peptides containing sequencesderived from the N-terminus of influenza virus hemagglutinin HA-2subunit in DNA/transferrin-polylysine complexes has been found tosubstantially augment the transferrin-polylysine mediated gene transfer(Examples 13 and 14).

Similar DNA combination complexes containing the tetra-antennarygalactose ligand-polylysine conjugate and the polylysine-modifiedinfluenza peptide InflupL (prepared as described in Example 6 or 13,respectively) have been prepared by adding the ligand-polylysineconjugate to plasmid DNA pCMVL to neutralize half of the DNA charge, theremainder of the charge being used to load the complexes with influenzapeptide-polylysine conjugate. The delivery of these DNA complexes,containing the synthetic ligand (gal)4, to BNL CL2 hepatocytes(transfections were carried out as described in Example 6 g) resulted ina luciferase gene expression (FIG. 43) that was significantly higherthan the expression obtained with transferrin as ligand. The expressionwas more than 500-fold higher than in control experiments obtained withDNA complexes lacking the influenza peptides, but containing the sameamount of polylysine (FIG. 43). The activity obtained with the DNAcombination complexes was also approx. 30-fold higher than withDNA/(gal)4 pL complexes incubated with cells in the presence ofchloroquine.

b) Ternary complexes containing adenovirus conjugate

Complexes were prepared as follows: biotinylated adenovirus d1312(prepared as in Example 19; 2 μl, 6 μl or 18 μl; 10¹² particles/ml) in50 μl HBS were mixed with streptavidin-polylysine (100 ng, 160 ng, or480 ng) in 100 μl HBS; after a 30 min incubation, a solution of 6 μgpCMV-L in 200 μl HBS was added, and after further 30 min, a solution of3.8 kg (gal)4 pL (prepared as in Example 6) or 7 μg TfpL in 150 μl HBSwas added.

The DNA complex solutions were added to each 300,000 cells (ATCC TIB73,ATCC TIB74, ATCC TIB75, ATCC TIB76) grown in 6 cm plates in high glucoseDMEM+2%FCS. Further cell culture procedures and luciferase assays wereperformed as described. Gene expression (after 24 h) as shown in FIG.44.

Example 29 DNA Transfer With Transferrin-polylysine in the Presence ofFlee and Conjugated Rhinovirus

a) Rhinovirus HRV-2 preparations

Rhinovirus HRV-2 was prepared and purified as described (Skern et at.,1984; the disclosure of which is fully incorporated by referenceherein).

A 400 μl solution of rhinovirus (approx. 30 μg) in HBS (150 mM NaCl/5 mMHEPES, pH 7.9)/10% glycerol was treated with 10 nmol of NHS-LC-biotin(Pierce 21335). After incubation for 3 hrs at room temperature, thevirus was separated from unincorporated biotin by extensive dialysisagainst HBS/40% glycerol at 4° C.

Light-sensitive rhinovirus, prepared by growing the virus in thepresence of acridine orange, was inactivated as described (Madshus etal., 1984).

b) Preparation of DNA complexes and transfections

i) Transferrin-polylysine I DNA complexes were prepared by mixing asolution of 6 μg of plasmid DNA pCMVL in 330 μl HBS (150 mM NaCl, 20 mMHEPES, pH 7.3) with a solution of 8 μg TfpL290 in 170 μl HBS.

DNA complexes were mixed with 1.5 ml of medium (DMEM plus 2%FCS) andwith 0.14 μg to 3.5 μg rhinovirus HRV-2 (or inactivated HRV-2). Themixture was added to NIH 3T3 cells (300,000 cells per 6 cm plate). Fourhours later the transfection medium was replaced by fresh 4 ml of DMEMplus 10% FCS, Cells were harvested after 24 hrs and assayed forluciferase activity as previously described (FIG. 45A).

ii) DNA combination complexes containing transferrin-polylysine andrhinovirus-polylysine conjugates were prepared as follows: a 100 μlsolution of biotinylated rhinovirus HRV-2 (3.5 μg) in HBS was mixed with1 μg streptavidinylated polylysine in 100 μl HBS. (ale other virusconcentrations were mixed with appropriate portions.) After 30 min atroom temperature, the solution was mixed with 6 μg of plasmid DNA in 150μl HBS, incubated for a further 30 min at room temperature, andsubsequently mixed with 6 μg TfpL290 in 150 μμl HBS.

DNA complexes were mixed with 1.5 ml of medium (DMEM plus 2%FCS) andadded to NIH 3 M cells (300,000 cells per 6 cm plate). Further treatmentof the cultures and the assay for luciferase activity was performed asdescribed in i) (FIG. 45B).

Example 30 Transfection of HeLa Cells With Combination ComplexesContaining Ionically Bound Adenovirus

Complex formation A) DNA complexes were prepared by first mixing 30 μladenovirus d1312 (approx. 10⁹ PFUs) with 1 μg polylysine pLys450 (withan average chain length of 450 monomers) in 170 μl HBS and, after 30 minat room temperature, subsequent mixing with 6 μg of pCMVL-DNA in 170 μlHBS. After an incubation for another 30 min, complexes were mixed with 9μg TfpL190 in 170 μl HBS. An aliquot of the complex mixture (10%=50 μlsolution, 600 ng DNA; or 1%=5 μl solution 60 ng DNA) was diluted in 1.5ml DMEM plus 2% FCS and added to 300000 HeLa cells. After 4 h, 2 ml ofDMEM plus 20% FCS was added. Harvesting of cells 24 h after transfectionand luciferase assay were performed as described. The luciferaseactivity corresponding to the total extract were 29115000 light units(in the case of 600 ng DNA) and 1090000 light units (in the case of 60ng DNA).

Control experiment: Complex formation B) DNA complexes were prepared byfirst mixing 6 μg of pCMVL/DNA in 170 μl HBS with 1 μg polylysinepLys450 (with an average chain length of 450 monomers) in 170 μl HBSand, after 30 min at room temperature, subsequent mixing with 9 μgTfpL190 in 170 μl HBS. After an incubation for another 30 min, complexeswere mixed with 30 μl adenovirus d1312 (approx. 10⁹ PFUs). An aliquot ofthe complex mixture (10%=50 μl solution, 600 ng DNA; or 1%=5 μlsolution, 60 ng DNA) was diluted in 1.5 ml DMEM plus 2% FCS and added to300000 HeLa cells. After 4 h, 2 ml of DMEM plus 20% FCS was added.Harvesting of cells 24 h after transfection and luciferase assay wereperformed as described as in the previous examples. The luciferaseactivity corresponding to the total extract were 405000 light units (inthe case of 600 ng DNA) and 200 light units (in the case of 60 ng DNA).

Example 31 Local Application of DNA/adenovirus/transferrin-polylysineConjugates Into Rat Liver

a) Direct injection

The complexes were prepared as described in Example 19. They comprised200 μl biotinylated Adenovirus (d1312, 6.4 μg streptavidin-polylysine,48 μg pCMVL and 48 μg TfpL290 in a total volume of 2000 μl HBS. A maleSprague-Dawley rat of 240 g was anesthetized with Avertin and alaparotomy of 4 cm performed. The complex solution was injected into theleft lob of the liver. Then the wound was closed in layers. 48 hoursafter injection of the complexes the rat was sacrificed and theluciferase expression measured. In the area of injection 5615 lightunits/per mg protein of the liver homogenate were measured. Totalluciferase activity at the injection site was 370,600 light units.

b) Application of conjugates to the liver via the bile draining system

The complexes were prepared as follows: 200 μμl biotinylated Adenovirusis d1312 diluted with 200 μl HBS were incubated with 6.4 μgstreptavidin-modified polylysine in 400 μl HBS for 30 minutes at roomtemperature. Then 48 μg of pCMV-L in 800 μl HBS were added. After 30minutes of incubation 48 μg of TfpL in 900 μμl HBS were further added.For application of the complexes male Sprague Dawley rats (250 g bodyweight) were anesthetize with Avertin and the abdomen opened with amedian incision. The intestine was displaced to the left side of thebody and a 27 G needle, which had been attached to a tube and a I mlsyringe was inserted into the bile duct. The injection of the complexeswas performed over a period of 4 minutes. Then the needle was retractedfrom the bile duct and the injection site sealed with a fibrin sealer(Immuno). The abdominal wound was closed With sutures and metal clips.After 30 hours the rat was killed and samples from different lobes ofthe river were assayed for luciferase gene expression. The pea activityof luciferase was 19000 light units/mg protein and the calculatedoverall expression in the total liver was in the range of 2.7×10⁶ lightunits.

Example 32 Local Application of DNA/adenovirus/transferrin-polylysineConjugates Into the Clamped Mouse Tail Vein

The complexes were prepared as described in Example 19. They comprised45 μl biotinylated Adenovirus d1312, 0.8 μg streptavidin-polylysine, 6μg pCMVL and 24 μg TfpL290 in a total volume of 150 μl HBS. Thecomplexes were injected into the tail vein of a male C3H/He mouse (twomonths old), which had been anesthetized with Avertin. Immediately afterinjection the tail vein was clamped for 20 minutes at the proximal anddistal end of the tail such that the complex solution was restricted tothe segment of the tail vein which had been injected and could not beflushed by the blood. 48 hours after injection the mouse was sacrificedand the tail vein prepared. Luciferase expression was measured in thehomogenate of the tail vein segment. Expression resulted in 2,600 lightunits/3 cm tail vein.

Example 33 Transfection of Primary Human Melanoma Cells

Primary melanoma cells were isolated from a melanoma, which had beensurgically removed from a patient, by mechanically disrupting the tumorin RPMI 1640 medium plus 5% FCS, 2mM glutamine, and antibiotics andpressing the tissue fragments through a steel sieve. The tumor cellswere washed several times by centrifugation and subsequent resuspensionand seeded into 725 cell culture flasks. 24 hours after isolation, thetumor cells were transfected with combination complexes comprising 3 μl,9 μl or 27 μl biotinylated Adenovirus d1312 (1×10¹² virus/ml), 0.5 μgstreptavidin-polylysine, 6 μg pCMVL and 7 μg TfpL290 in a total volumeof 500 μl HBS. 36 hours after transfection the cells were harvested andthe expression of luciferase determined. Results are shown in FIG. 46.

Example 33B Transfection of Primary Human Melamona Cells WithLDL-polylysine Conjugates

Preparation of LDL polylysine 300 conjugates. A solution of 10 mg (14.3μmole) LDL (Low Density Lipoprotein, Sigma, L-2139, molecular weight3,500,000, particle size approx. 26 nm) in 2 ml HBS was mixed with 143μl of a 10 mM ethanolic solution of SPDP (1.43 μmole; Pharmacia) andallowed to react for 2 h at room temperature. Gel filtration over aSephadex G-25 column (14×140 mm) with HBS yielded 3.2 ml of a solutionof approx. 10 mg LDL, modified with 0.70 μmole pyridyldithiopropionateresidues. The solution was mixed with 1.2 ml 5 M NaCl in order toprevent precipitation that would otherwise occur upon addition ofpolylysine. Poly(L)lysine 300 was modified with SPDP as described andtreated with dithiothreitol and subsequent gel filtration in order toobtain the mercapto modified form. The above modified LDL solution wasmixed with a solution of 0.33 μmole polylysine 300, modified with 0.76μmole mercapto groups, in 4 ml 2 M NaCl, 0.5 M HEPES pH 7.9, under argonand allowed to stand for 48 h at room temperature. The reaction solutionwas diluted to 32 ml with sterile water (NaCl concentration approx. 0.5M) and separated by ion exchange chromatography (Biorad Macroprep S,10×100 mm, 20 mM HEPES pH 7.3, gradient 0.2×3 M NaCl). The productfractions eluted at salt concentrations of 1.8 M-2.2 M and were pooled.After dialysis against HBS conjugates consisting of 2.35 mg (3.4 nmole)LDL, modified with 190 nmole polylysine (corresponding to 7.5 mg of freepolylysine in its basic form) were obtained. This corresponds to anaverage modification of the LDL particles with approx. 55 polylysinechains.

Preparation of DNA complexes 18 μl biotinylated adenovirus d1312preparation were diluted with HBS to 100 μl. 1.2 μgstreptavidin-polylysine were adjusted with HBS to 100 μl and mixed withthe above solution. After 30 min, 150 μI HBS containing 6 μg pCMVL wereadded. After further 30 min 300 μl HBS containing 4 μg LDLpL (content ofpolylysine: 20 μg) were added. The obtained solution was mixed with 1.5ml DMEM +10% FCS and added to 3×10⁵ primary melanoma cells in a 6 mmculture dish (designated HMM1 and HMM4) which were prepared as describedin a). The further cell culture procedure and the luciferase assay wereperformed as described. The results are given in FIG. 47.”

Example 34 Transfection of Primary Human Fibroblasts

Human skin biopsies were put into a 6 cm petri dish containing DMEM, 2mM glutamine, 20% FCS and antibiotics. Then the biopsies were thoroughlyminced with a surgical knife and cultured in the presence of 3 ml mediumfor 5 days. Thereafter the cells were washed with fresh DMEM containing2 mM glutamine 10% FCS and antibiotics and cultured for further 7 days.After this period of time the cells were trypsinized and subculturedinto new petri dishes. When the cells were almost confluent, they weretrypsinized again and stored frozen until transfection. For transfectionthe cells were thawed and seeded into 6 cm petri dishes and cultured inDMEM containing 2 mM glutamine 10% FCS and antibiotics. The transfectionconjugates were prepared as follows: 3 μl, 10 μl, 20 μl and 30 μl ofbiotinylated adenovirus d1312 were incubated with 0.1 μg, 0.3 μg, 0.5 μgand 0.8 μg polylysine-modified streptavidin in 150 μl HBS for 30 minutesat room temperature. Then 6 μg of pCMV-βgal plasmid in 170 μl HBS wereadded and the mixture was incubated for further 30 minutes. In the finalstep 7.8 μg TfpL for the conjugates with 3 μl d1312, 7 μg TfpL for 10 μld1312 and 6 μg TfpL for the conjugates with 20 μl and 30 μl d1312 in 170μg HBS were added. After an incubation period of 30 minutes theconjugates were applied to the cells in 2 ml DMEM containing 2 mMglutamine, 2% FCS and antibiotics and the cells were incubated for 4hours at 37° C. Then the medium was removed and culture was continued at37° C. with DMEM containing 2 mM glutamine, 10% FCS and antibiotics.After 48 hours the expression of β-galactosidase was demonstrated asdescribed in previous Examples.

In the transfection with 3 μl d1312 14% of the cells revealed productionof ,β-galactosidase, with 10 μl d1312 32% positive cells were obtained,with 20 μl d1312 39% and with 30 μl d1312 64% of the cells werepositive.

Example 35 Gene Transfer Using Non-viral Endosomolytic Agents

a) Synthesis of membrane disrupting peptides

i) Peptide synthesis:

Peptides were synthesized on an automatic synthesizer (ABI 431A) by thesolid phase method using p-alkoxybenzylalcohol resin (0.97 mmol/g) assolid support and Fmoc-protected amino acids. The carboxy-terminal aminoacid was coupled to the resin via the symmetric anhydride. Subsequentamino acids were coupled by the 1-hydroxybenzotriazoledicyclohexylcarbodiimide method. The following side chain protectinggroups were used: (Trt)Asn, (Trt)Cys [(t-Bu)Cys in case of EALA andGLF], (t-Bu)Glu, (Trt)His, (t-Bu)Ser.

EALA: (SEQ ID NO:5) Tip Glu Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala LeuAla Glu His Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala GlyGly Ser Cys

GLF (SEQ ID NO:6) Gly Leu Phe Gly Ala Leu Ala Glu Ala Leu Ala Glu AlaLeu Ala Glu His Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala AlaGly Gly Ser Cys

GLF-II (SEQ ID NO:7) Gly Leu Phe Gly Ala Leu Ala Glu Ala Leu Ala Glu AlaLeu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala AlaGly Gly Ser Cys

GLF-delta (SEQ ID NO:8) Gly Leu Phe Glu Leu Ala Glu Ala Leu Ala Glu AlaLeu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala AlaGly Gly Ser Cys

EALA-Inf (SEQ ID NO:9) Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu AsnGly Tip Glu Gly Au Ala Glu Ala Ala Ala Glu Ala Leu Glu Ala Leu Ala AlaGly Gly Ser Cys

EALA-P50 (SED ID NO: 10) Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu AsnGly Tip Glu Gly Leu Ala Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala AlaGly Gly Ser Cys

P50 (SED ID NO:11) Gly Leu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn GlyTrp Glu Gly Met Ile Asp Gly Gly Gly Cys

The peptides were cleaved from the resin and the side chain protectinggroups were removed [except (t-Bu)Cys] by treatment of 100 mgpeptide-loaded support with 3 ml of a mixturephenol/ethanedithiol/thioanisol/ water/trifluoroacetic acid0.75:0.25:0.5:0.5:10 for 1.5 h at room temperature. The crude peptideswere precipitated in ether and washed two times. The S-t-Bu protectedpeptides EALA and GLF were dissolved in a small volume 1 Mtriethylammonium bicarbonate (TEAB) pH 8, diluted to 100 mM TEAB andfurther purified by reverse phase HPLC on a Nucleosil 500-5C4 column(0.1% TFA-acetonitrile gradient). Both peptides eluted at about 50%acetonitrile. The free Cys-SH form of the peptides was obtained bydeprotecting Trt-Cys peptides in the same way as described above. Thecrude peptides (5 mg) were dissolved in 100 μl 100 mM TEAB, pH 8,containing 1 μl -mercaptoethanol and purified by gel filtration(Sephadex G-25, 100 mM TEAB, 0.5 mM EDTA) and freeze drying orion-exchange chromatography (Mono Q Pharmacia, 20 mM Hepes, pH 7.3,gradient 0 to 3 M NaCl, the peptide elutes at 1.5 M NaCl).

ii) Modification with N-(hydroxyethyl)maleimide

The C-terminal SH group of the peptides GLFdelta, GLF-II, EALA-Inf,EALA-P50, P50 was blocked after gel filtration of the free SH form(Sephadex G-25, 20 mM Hepes, pH 7.3, 0.5 ml EDTA) by reaction with a1.3- to 10-fold molar excess of N-(hydroxyethyl)maleimide (1 h, roomtemperature). Excess maleimide was removed by gel filtration (SephadexG-25, 100 mM TEAB, pH 8) and the peptides (GLF-delta-mal, GLF-II-mal,EALA-Inf-mal, EALA-P50-mal, P50-mal) were obtained as triethylammoniumsalt upon freeze drying.

iii) Modification with 2,2′-dithiobispyridine:

The free SH peptides were reacted with 10 equivalents of2,2′-dithiobispyridine (20 mM Hepes, pH 7.9, 0.5 mM EDTA) over night atroom temperature. Excess reagent was removed by gel filtration (SephadexG-25, 100 mM TEAB, pH 8) or ion-exchange chromatography (Mono QPharmacia, 20 mM Hepes, pH 7.3, gradient 0 to 3 M NaCl, the peptideelutes at 1.5 M NaCl) to obtain the (2-pyridylthio)-Cys peptides(GLF-delta-SSPy, GLF-II-SSPy, EALA-Inf-SSPy, 31EAIA-P50-SSPy, P50-SSPy).

iv) Dimerization of peptides:

The homodimer of P50 (P50 dim) was prepared by reacting equimolaramounts of P50Cys-(2-pyridylthio) and P50-Cys-SH in 20 mM Hepes, pH 7.3,over three days at room temperature. The reaction mixture was separateda Mono Q column (HR-515 Pharmacia; 20 mM Hepes, pH 7.3, gradient 0.09 Mto 3 M NaCl, P50-dimer was eluted at 1.1 M NaCl). The heterodimerGLF-SS-P50 was prepared analogously by reaction of peptide P50, freemercapto form, with pyridylthio-modified peptide GLY.

b) Liposome leakage assay:

The ability of the synthetic peptides to disrupt liposomes was assayedby the release of fluorescent dye from liposomes loaded with aself-quenching concentration of calcein. Liposomes were prepared fromegg phosphatidylcholine by reverse phase evaporation (Szola andPapahadjopoulos, 1978) with an aqueous phase of 100 mM calcein, 375 mMNa⁺, 50 mM NaCl, pH 7.3 and extruded through a 100 nm polycarbonatefilter (MacDonald et al., 1991) to obtain a uniform size distribution.The liposomes were separated from unincorporated material by gelfiltration on Sephadex G25 with an iso-osmotic buffer (200 mM NaCl, 25mM Hepes, pH 7.3). For the leakage assay at various pH values, theliposome stock solution was diluted (6 μl/ml) in 2 x assay buffer (400mM NaCl, 40 mM Na citrate). An aliquot of 100 μl was added to 80 μl of aserial dilution of the peptide in water in a 96-well microtiter plate(final lipid concentration: 25 μM) and assayed for calcein fluorescenceat 600 nm (excitation 490 nm) on a microtiter-plate fluorescencephotometer after 30 min of incubation at room temperature. The value for100% leakage was obtained by addition of 1 μl of a 10% Triton X-100solution. The leakage units were calculated as reciprocal value of thepeptide concentration, where 50% leakage was observed (i.e. the volume(μl) of liposome solution which is lysed to 50% per μg of peptide).Values below 20 units are extrapolated. The results of the liposomeleakage assay are shown in FIG. 48. GLF and EALA exhibited the highestpH specific activity.

c) Erythrocyte lysis assay:

Fresh human erythrocytes were washed with HBS several times andresuspended in 2 x assay buffer of the appropriate pH (300 mM NaCl, 30mM Na citrate) at a concentration of 6.6 10⁷/ml. An aliquot of 75 μl wasadded to 75 μl of a serial dilution of the peptide in water in a 96-wellmicrotiter plate (cone type) and incubated for 1 h at 37° C. withconstant shaking. After removing of the unlysed erythrocytes bycentrifugation (1000 rcf, 5 min) 100 μl of the supernatant wastransferred to a new microtiter plate and hemoglobin absorption wasdetermined at 450 nm (background correction at 750 nm). 100% lysis wasdetermined by adding 1 μl of a 10% Triton X-100 solution prior tocentrifugation. The hemolytic units were calculated as reciprocal valueof the peptide concentration, where 50% leakage was observed (i.e. thevolume (μl) of erythrocyte solution which is lysed to 50% per μg ofpeptide). Values below 3 hemolytic units are extrapolated. The valuesare given in FIG. 49. As can be seen, P50 dim and EALA-P50 exhibited thehighest pH specific activity with regard to lysis of cells and/orrelease of larger molecules such as hemoglobin. The P50 monomers P50maland P50 SS-Py had lower activity. Melittin showed the highest activity,which was, however, not specific for acidic pH.

d) Preparation of DNA combination complexes:

DNA complexes were prepared by first mixing 6 μg of pCMVL-DNA in 150 μlHBS with 4 μg TfpL290 in 150 μl HBS and subsequent mixing with 4 to 20μg of poly(L)lysine290 in 100 μl HBS after 30 min at room temperature;after further 30 min incubation at room temperature, 0.3 to 30 μg ofpeptide in 100 μl HBS was added and incubated for another 30 min. Theoptimal amount of endosomolyic agent was determined in preliminarytitrations by assaying the resulting gene transfer efficiency (see Table3 for gene transfer to BNL CL.2 cells). Simultaneous addition of pLysand endosomolytic agent as well as the use of larger volumes for thecomplex preparation (1.5 ml final volume) was shown to give comparable(or better) transfection efficiencies. In these experiments, thenon-peptidic amphipathic substances desoxycholic acid and oleic acidwere also shown to augment DNA delivery.

e) Transfection of cells:

Adherent cell lines (BNL CL.2 hepatocytes or NIH 3T3 cells,respectively) were grown in 6 cm dishes for 1 to 2 days prior totransfection (DMEM medium with 10% FCS; 300,000 cells per dish). Themedium was removed and 1.5 ml of DMEM (2% FCS) and 500 μl of the DNAcomplexes were added. Alternatively, 0.5 ml DMEM 6% FCS and 1.5 ml ofDNA complexes was used. After 4 h incubation 2 ml DMEM (18% FCS) wasadded. Alternatively, the transfection medium can be replaced by 4 ml ofDMEM with 10% FCS. Harvesting of cells and luciferase assays wereperformed 24 h after transfection as described previously. The lightunit values shown, represent the total luciferase activity of thetransfected cells.

Transfection of BNL CL.2 hepatocytes is shown in FIG. 50:

FIG. 50A) DNA complexes were prepared by first mixing 6 μg of pCMVL-DNAin 250 μl HBS with 4 μg TfpL290 in 250 μl HBS and subsequent mixing with20 μg of poly(L)lysinem90 in 750 μl HBS after 30 min at roomtemperature; after further 30 min incubation at room temperature,indicated amounts of peptides in 250 μl HBS were added. After anincubation for another 30 min, complexes were mixed with 0.5 ml DMEMplus 6% FCS and added to 450,000 cells.

FIG. 50B) DNA complexes were prepared as follows. A solution of 6 μg ofpCMVL-DNA in 500 μl HBS was mixed with 4 μg TfpL290 in 250 μl HBS andleft for 30 min at room temperature. A 500 μl solution of 20 μg ofpoly(L)lysine₂₉₀ in HBS with mixed with indicated amounts of peptides in250 μl HBS and immediately added to the TfpL/DNA mixture. After further30 min incubation at room temperature the complexes were mixed with 0.5ml DMEM plus 6% FCS and added to 450,000 cells. Harvesting of cells 24 hafter transfection and luciferase assays were performed as describedpreviously.

The experiments carried out with NIH3T3 cells are shown in FIG. 51. Thepreparation of complexes according to A) and B) was the same as for thetransfection of TIB 73.

In the cell culture experiments P50 dim and EALA-P50 exhibited thehighest activity, EALA and GLF had medium activity, whereas P50 monomersand melittin had low activity.

Example 36 Gene Transfer Using a Synthetic Non-viral Peptide With anOligolysine C-terminal Extension

A peptide with the sequence (SEQ ID NO:4) Met Ala Gln Asp lie Ile SerThr Ile Gly Asp Leu Val Lys Tip Ile Ile Asp Thr Val Asn Lys Phe Thr LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys was synthesized and purifiedaccording to the method described in the previous Example. This peptideis derived from the δ-toxin of Staphylococcus aureus (SEQ ID NO:3) MetAla Gln Asp He Ile Ser Thr Ile Gly Asp Leu Val Lys Tip Ile Ile Asp ThrVal Asn Lys Phe Thr Lys Lys, which is known to possess specificity formembrane disruption at acidic pH (Thiaudiere et a1., 1991; Alouf et al.,1989), by extension by additional 10 lysine residues.

DNA complexes were prepared by first mixing 6 μg of pCMVL-DNA in 170 μlHBS with 4 μg TfpL290 in 170 μl HBS and subsequent mixing withapproximately 3 μg of peptide in 170 μl HBS after 30 min at roomtemperature. After an incubation of another 30 min, complexes were mixedwith 1.5 ml DMEM plus 2% FCS and added to 450,000 BNL CL.2 hepatocytes.After 2 h, 2 ml of DMEM plus 20% FCS were added. Harvesting of cells 24h after transfection and measuring luciferase activity were performed asdescribed in the previous Examples. The luciferase activitycorresponding to the total extract was 481,000 light units.

Example 37 Transfection of Hepatocytes in the Presence ofMelittin-peptides With a C-terminal Oligo-Lys-tail

Peptides of the sequences (N to C terminus) (SEQ ID NO: 12) Gly Ile GlyAla Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu He Ser Trp Be LysArg Lys Arg Gln Gin Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys (designatedmel 1) and (SEQ ID NO: 13) Gly lie Gly Ala Val Leu Glu Val Leu Glu ThrGly Leu Pro Ala Leu Ile Ser Trp lie Lys Arg Lys Arg Gln Gin Lys Lys LysLys Lys Lys Lys Lys Lys Lys (acidic mutant, designated mel 2) weresynthesized as described in Example 36.

DNA complexes were prepared by first mixing 6 μg of pCMVL-DNA in 170 μlHBS with 4 μg TfpL290 in 170 μl HBS and subsequent mixing withapproximately 3 μg of peptide mell or 5 μg of mel2 in 170 μl HBS after30 min at room temperature. After an incubation for another 30 min,complexes were mixed with 1.5 ml DMEM plus 2% FCS and added to 450000BNL CL.2 cells, cultivated as described in Example 36. After 4 h, 2 mlof DMEM plus 20% FCS was added. Harvesting of cells 24 h aftertransfection and luciferase assay were performed as described. Theluciferase activity corresponding to the total extract were 9200 lightunits (in the case of mel1) and 9400 light units (in the case of mel2).

Example 38 Interferon Alpha Expression in HeLa Cells

HeLa cells (5×10⁵ cells per 6 cm dish) were transfected withpAD-CMV1-IFN encoding human interferon alpha2c under the control of theCMV enhancer/promoter (described in DE 40 21 917 A. pAD-CMV1-IFN wasobtained by recloning the HindIII-XbaI IFN-∝2c insert into pAD-CMV1).Samples of 6 μg DNA in 330 μl HBS were mixed with 8 μg of TfpL in 330 μlHBS and allowed to assemble for 30 minutes at room temperature. Samples6-10 contained only 4 μg of TfpL and after the first 30 minutesincubation an aliquot of P16pL (20 μg) in 160 μl of HBS was added toboth samples 6 and 7 and an aliquot of pLys 290 (20 μg) was added tosamples 8, 9 and 10. After an additional 30 minute incubation, aliquotsof 160 μl of HBS containing 10 μl (sample 8) or 50 Al (sample 9 and 10)of free P16 were added (for synthesis of P16 and P16pL, see Example 13).After an additional 30 minute incubation, the samples were supplied toHeLa cells in 2 ml of DMEM/2%FCS in the presence of the followingadditional compounds. Sample 2, 7 and 10 contained 100 μM chloroquine,samples 3 and 4 contained 5 and 15 μl of adenovirus d1312, (1×10¹²particles/ml), sample 5 contained 15 μl of the same virus,psoralen-inactivated. As controls for adenovirus stimulation ofendogenous interferon production, samples 11, 12 and 13 were treatedwith aliquots of virus equal to samples 3, 4 and 5). At 2 hours aftertransfection 5 ml of fresh DMEM+10%FCS were added to the cells. At 48hours after transfection the medium was removed and replaced with 2 mlof fresh DMEM+10% FCS. This medium was harvested at 72 hours posttransfection and an ELISA analysis for interferon alpha was performed asdescribed in DE 40 21 917. The interferon alpha levels (in ng/ml) aredisplayed in FIG. 52.

TfpL functioned poorly to deliver IFN genes to these cells, consistentwith the previous observations using luciferase or β-gal reporter genes.The presence of chloroquine generated a detectable signal (ca. 7 ng/ml,sample 2), but adenovirus d1312 stimulated DNA delivery in a dosedependent fashion (samples 3 and 4). Treating these cells withcomparable quantities of virus in the absence of IFN DNA complexes didnot result in a detectable interferon signal (samples 11 and 12).Transfection with the synthetic influenza derived endosomolytic peptideP16(see Example 13) as a conjugate (sample 6,7) or as a peptideionically bound to the surface of TfpL/DNA complexes (samples 8, 9 and10, for binding of peptides see Example 35) generated detectable levelsof interferon production, which was enhanced with the peptide conjugatein the presence of chloroquine (sample 7).

Example 39 Gene Transfer Into B-lymphoblastoid Cells

Human-Ig polylysine conjugates and anti-human-Ig polylysine conjugateswere prepared in the following manner (coupling was carried out withmethods known in the literature (Jung et al., 1981) by introducingdisulfide bridges after modification with succinimidyl-pyridyldithiopropionate).

a) Preparation of anti-human-Ig/polylysine 300 conjugates.

A solution of 2 mg goat anti-human-Ig (Southern BiotechnologyAssociates, Inc., Birmingham, AL, USA) in HBS (150 mM NaCl, 20 mM HEPES,pH 8.7) was mixed with 14 μl of 5 mM ethanolic solution of SPDP(Pharmacia). After 10 h at room temperature, the solution was filteredon a Sephadex G 25 column ((eluted with 100 mM HEPES, pH 7.3), yielding1.3 mg of anti-human-Ig, modified with 30 nmole pyridylthiopropionateresidues. Poly(L)lysine 300 (Sigma; average degree of polymerization 300lysine residues) was modified analogously and brought into themercapto-modified form by treating with dithiothretol and subsequent gelfiltration. A solution of 12 nmole polylysine 300, modified with 29nmole mercapto groups in 0.3 ml HBS was mixed with the aboveanti-human-Ig under exclusion of oxygen and allowed to stand overnightat room temperature. By addition of 5 M NaCl, the reaction mixture wasadjusted to 0.6 M NaCl. The conjugates were isolated by ion exchangechromatography Pharmacia, Mono S HR 515); after dialysis against 25 mMHEPES, pH 7.3, conjugates comprising 0.33 mg anti-human-Ig, modifiedwith 4 nmole polylysine 300 (molar ratio 1:2) were obtained.

b) Preparation of human-IgG/polylysine 300 conjugates.

A solution of 19.5 mg (122 nmole) antibody (Sigma 14506) in 2 ml HBS wasmixed with 39 μl of 10 mM ethanolic solution of SDP. After 2.5 h at roomtemperature, the solution was passed over a gel column (Sephadex G25;eluted with 100 mM HEPES, pH 7.9) yielding 19 mg (119 nmole) human-IgGmodified with 252 nmole pyridyldithiopropionate residues. Polylysine 300was treated analogously to a) in order to obtain the mercapto form. Asolution of 119 nmole polylysine 300 modified with 282 nmole mercaptogroups in 1 ml HBS was mixed with the above modified humanIgG underexclusion of oxygen and allowed to stand over night at room temperature.By addition of 5 M NaCl, the reaction mixture was adjusted to 0.6 MNaCl. The conjugates were isolated by ion exchange chromatography(Pharmacia, Mono S, 50 mM HEPES, pH 7.3; salt gradient 0.6 M to 3 MNaCl); : after dialysis against HBS , pH 7.3, conjugates comprising 9 mg(57 nmole) human-IgG, modified with 90 nmole polylysine 300 (molarration 1:1.6) were obtained.

c) Complex formation and transfection of cells.

Complexes were prepared as follows: Biotinylated adenovirus d1312 (30μl, 10¹² particles/ml) in 50 μl HBS were mixed withstreptavidin-polylysine (800 ng) in 100 μl HBS. After a 30 minincubation, a solution of μg pCMVL in 200 μl HBS was added and after afurther 30 min, a solution of 5.1 μg polylysine 450, 10.2 μg TfpL, 12 μghuman-Ig/polylysine conjugate or 10 μg anti-human-Ig/polylysineconjugate, in 150 μl HBS, were added. The DNA complex solutions wereadded to each 10⁶ B-lymphoblastoid cells (generated from humanperipheral blood mononuclear cells by immortalizing with Epstein Barrvirus as described by Wells and Crawford, 1989), grown in 24-well platesin ml of RPMI 1640+2% cell culture procedures and luciferase assays wereperformed as described. Gene expression after 48 h is shown in FIG. 53.

Example 40 Direct In Vivo Gene Transfer to Airway Epithelium EmployingAdenovirus-polylysine-DNA Complexes

In the present example, the direct in vivo gene transfer to therespiratory epithelium is accomplished in a rodent model usingadenovirus-polylysine-DNA complexes. This establishes the feasibility ofthis approach as a method to accomplish transient gene expression in therespiratory epithelium. The capacity to achieve genetic modification ofthe airway epithelial cells in situ offers a potential strategy toaccomplish gene therapy for disorders afflicting the airway epithelium.

The firefly luciferase reporter gene containing plasmid pCLuc4 was usedto form conjugate-DNA complexes which were delivered to cotton rats viainjection by the intratracheal route. The complexes (hTfpL/bAdpL) wereformed with human transferrin-polylysine and adenovirus that had beeninactivated by genomic deletion and treatment with psoralen plusUV-irradiation. This modification allows prolonged in vitro expressionconsequent to minimized adenoviral replication and/or gene expression.Lungs were harvested and lysates evaluated for luciferase geneexpression at various time points post-injection.

FIG. 54 shows the time course of heterologous gene expression in cottonrat airway epithelium transduced with humantransferrin-adenovirus-polylysine-DNA complexes. Ordinate representsluciferase gene expression as Light Units per 1250 μg total proteinderived from lung lysates. Experiments were performed 3-4 times each andresults expressed as mean+SEM. Maximum gene expression was noted at 24hr post-administration. There was a rapid decrease of net geneexpression such that levels diminished to background by day 7.

As shown in FIG. 54, the detectable in vivo gene expression mediated bythe adenovirus-polylysine DNA complexes was of a transient nature. Thisclosely parallels the expression pattern noted after lipofectin-mediatedin vivo gene transfer to the respiratory epithelium (Hazinski et al.,1991). This result is not unanticipated as the delivered DNA would bepresent as a plasmid episome lacking replicative or integrative capacity(Wilson et al.). In the present design the conjugate system lacks amechanism to mediate integration and thus the stable transductionfrequency would be expected to be low. Alternatively, attrition of themodified cells could explain the extinction of gene transfer in thelung. In terms of the observed transduction frequency, it ishypothesized that the low percentage of cells transduced in vivo couldrepresent a problem of initial binding of the vector to the targetairway epithelial cell. In this regard, differentiated airway epitheliummay present a vastly different cell surface receptor population thanthat characterizing immortalized airway epithelial cell lines or primarycultures of airway epithelial cells. Thus, vectors possessing a ligandwith a high binding affinity to the differentiated airway epithelium mayovercome this problem. In addition, various factors present in theenvironment of the airway epithelium may be deleterious tovector-mediated gene transfer. These factors include ciliary motion, thebronchial mucus, surfactant, and epithelial lining fluid proteases. Toevaluate these possibilities, various maneuvers may be employed tomodify the airway epithelial environment prior to vector delivery. Thesemanipulations include: 1) the paralysis of ciliary motion by lowtemperature or topical anesthesia; 2) dispersal of bronchial mucusthrough the use of mucolytic agents such as N-acetyl cysteine; and 3)the delivery of inhibitors of proteases to the epithelial lining fluidsuch as α₁-antitrypsin (α1AT) and bronchial protease inhibitor (BIP orSLIP). These various pre-delivery maneuvers can be non-invasivelyinstituted to attempt to mitigate potential in situ barriers to in vivoairway epithelial gene transfer.

In terms of the time course of heterologous gene expression in thetransduced animals, the rapid extinction observed could representeither: 1) loss of DNA from transduced cells or 2) loss of transducedcells secondary to vector-related toxicity. If vector toxicity is thebasis of the limited gene expression noted in vivo, this may beaddressed by the introduction of additional deletions into the genome ofthe adenovirus component of the vector.

Example 41 In Vivo Gene Transfer to Airway Epithelium EmployingMolecular Conjugate Vectors

Molecular conjugates with the capacity to bind selectively to theciliated airway epithelial subset have been derived utilizing ligandswith known specificity for this cellular target. The construction ofthese conjugate vectors entails: 1) the confirmation of the bindingproperties of the candidate ligands in the conjugate confirmation; and2) the addition of components to enhance internalization aftercell-specific binding. The candidate ligands include: 1) influenzavirus; 2) the influenza hemagglutinin (HA) glycoprotein; and 3) thelectin SNA (see Piazza, et at., 1991; and Baum, et al., 1990). Theseagents have been demonstrated to bind selectively to ciliated airwayepithelial cells. To evaluate the binding of the ligands in theconjugate confirmation, intact airway epithelial tissue was required asimmortalized cell lines and primary airway epithelial cultures lackedthe target surface markers characterizing the differentiated intactairway epithelium. Immunohistologic localization of binding to fixedhuman tracheal sections was employed to establish the cell-specificbinding properties of the candidate ligands, and thus this assay systemwas used to evaluate the binding properties of the correspondingderivative conjugate vectors.

The ligand and polylysine components of the SNA-polylysine conjugateswere complexed via a biotin-avidin bridge. Biotin SNA (3.7 μg) wasdiluted in 175 μl of HBS and combined with streptavidin-polylysine (1.8μg), which had previously been diluted in 175 μl of HBS. The abovemixture was incubated at room temperature for 30 min prior to theaddition of 6 μg of pRSVL diluted in 150 μl of HBS followed by another30 min incubation at room temperature to allow the complexes to form.Human transferrin-polylysine conjugates consisting of human transferrincovalently linked to poly(L)lysine, hTfpL, were prepared as describedherein. Conjugate-DNA complexes were prepared by dilution of 6 μg ofpRSVL in 350 μl HBS followed by addition of 8 μg of hTfpL diluted in 150μl of HBS. Complexes were allowed to form by incubating for 30 min atroom temperature. To form polylysine-DNA complexes, poly()lysine450,pL450, (3 μg) or pL295 (4 μg) was diluted in 150 μl of HBS, then addedto 6 μg of pRSVL DNA diluted in 350 μl of HBS. The two components wereincubated at room temperature for 30 min. To prepare a complex with twoligand moieties: SNA and hTf at a one-to-one ratio, the bSNA-StpLcomplex was first formed combining 1.9 μg of bSNA diluted in 175 μl ofHBS and 0.9 μg of StpL diluted in 175 μl of HBS. The complex was allowedto form at room temperature for 30 min. pRSVL (6 μg) diluted in 150 μlof HBS was then followed by another 30 min incubation period. hTfpL (4μg) diluted in 150 μl of HBS was added to further condense the DNA whileincorporating the second ligand. This step was again followed by a 30min incubation period at room temperature.

The basis of this assay was to administer conjugate-DNA complexes totracheal sections, followed by detection of conjugate binding with aprimary antibody. The primary antibody of choice was a monoclonalanti-transferrin antibody that was specific for the human transferrinligand found in the conjugate-DNA complexes. The signal of the primaryantibody was then amplified using an anti-mouse antibody containinghorseradish peroxidase. Positive binding was observed as intense redstaining. The assay was optimized using the SNApL-DNA complex Monoclonalanti-transferrin antibody was used to detect the human transferrinligand. A series of conjugate-DNA complexes were formed to determine thebinding specificity of the SNApL/hTfpL-DNA complex. Positive stainingwas observed in the ciliated subset of respiratory epithelial cells withthe SNApL/hTfpL DNA complex using the monoclonal anti-transferrinantibody to determine binding specificity.

To validate this result, the following controls were executed: (1)Tracheal tissue that had only been treated with HRS, the conjugatebuffer, showed no positive staining following incubation with primaryand secondary antibodies. This result eliminated the possibility ofcross-reactivity between either of the two antibodies and human trachealtissue. (2) Following incubation with the SNApL/hTfpL-DNA complex, thetissue section was treated with an irrelevant antibody, ananti-neumaminidase monoclonal antibody (PY203), to determine whether ornot the secondary antibody was binding directly to the conjugate-DNAcomplex and not the primary antibody. The lack of positive staining forthis control eliminated this possibility. (3) Tracheal tissues areincubated with pL-DNA and treated with both primary and secondaryantibodies to determine whether the primary antibody was binding to thepolylysine component of the conjugate. No positive staining wasobserved, eliminating this possibility. (4) A tissue section wasincubated with SNApL-DNA followed by treatment with primary andsecondary antibodies. A negative result for this control eliminated thepossibility of the primary antibody binding to the SNA component of theconjugate. (5) A tissue section was incubated with hTfpL-DNA todetermine whether or not the SNApL/hTfpL-DNA complex was binding byvirtue of the human transferrin ligand and/or the polylysine component.Red positive staining similar to that observed for the SNApL/hTfpL-DNAcomplex was observed (FIG. 55). These results indicate that theSNApL/hTfpL-DNA complex could be binding by virtue of the SNA ligand,the human transferrin ligand, or the polylysine component.

To determine whether or not the above conjugate is binding to ciliatedairway epithelial cells by virtue of its polylysine component, anirrelevant ligand-polylysine complex was constructed. This irrelevantligand was the antibody MP301, which possesses specificity for theMycoplasma P1 protein.

Antibody-polylysine MP301pL (1.9 ng/ml; 5.5 μg in 150 μl HBS) wascombined with a plasmid DNA pIB120 (6.0 μg in 350 μl HBS) and incubated30 min at room temperature to form antibody-polylysine-DNA complexes.The complex (50 μl) was applied to hydrogen peroxide (3%) treated, fixedhuman trachea for 1 hr at °C. Blocking serum was added followed by thelink antibody (biotinylated anti-mouse immunoglobulin) and incubatedwith peroxidase-labelled streptavidin. Staining was completed with AECsubstrate. Intense red staining was noted in the apical region ofciliated airway epithelial cells. This pattern was not different fromthat noted with hTfpL and SNA/hTfpL complexes. It was concluded thatnonspecific binding was occurring. This nonspecific binding was mostlikely due to polylysine interaction with the apical membrane glycocalyxconstituents.

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TABLE 1A Transfection Method TfpL TfpL + TfpL + TfpL + + free coupledInflu- CELL TYPE TfpL chloroquine AdenoV AdenoV pLys Complex: (A.) (A.)(B.) (E.) (F.) Hepatocytes Hep G2 − +/− +++ BNL.C12 − +/− ++++ +++++ +++primary − − − ++ Myoblasts/myotubes C2C12 +/− +/− +++++ G8 +/− +/− +++++primary +++ Fibroblasts 3T3 + ++ +++ +++++ ++ Mov-13 +/− +/− +++++ +mouse L-cells +/− + + +++++ ++ primary ++++ Endothelial cells pig aorta+/− + +++ +/− Human neuro- blastoma cell line GI-ME-N +/− +/− +++ ++++HeLa cells + + +++++ +++++ +++ Mouse ES cells CCE +/− + ++ Bruce 4 +/−++ +++ Erythroid cells K562 − ++++ + +++++ + chicken HD3 ++ ++++ ++++++++ Bone marrow mouse − − ++ chicken +/− + +++ EBV-transformed − − −+++ + human B-cells Mouse plasma cell + ++ +++ lines (MPC11.SP2/0) +/−Luciferase activity 1000-10,000 light units per 10⁶ cells + 10,000-1million light units ++ 1-5 million light units +++ 5-50 million lightunits ++++ 50-100 million light units +++++ >100 million light unitsTfpL transferrin-polylysine AdenoV replication-defective adenovirusd1312 Influ-pLys influenza HA-2 N-terminal peptide-polylysine

TABLE 2A Membrane-active Proteins Viral fusion proteins N-terminalfusion sequence enveloped viruses influenza virus myxoviridae HA2 pH 5White, 1990 und Takahashi, 1990 VSV rhabdoviridae G pH 5 Hoekstra, 1990vaccinia virus 14 kDa pH 5 Gong et al., 1990 mendai virusparainfluenzav. F1 pH 7 Hoekstra, 1990 und Gething et al., 1978 measlesvirus parainfluenzav. F pH 7 Hoekstra, 1990 HIV retroviridae gp41 pH 7Slepushkin et al., 1992 SIV retroviridae gp41 pH 7 Ruyaschaert undVandenbranden, 1991; Franchini, 1989 unenveloped viruses polio virusenteroviridae vp1 pH 5 Fricks und Hogie, 1990 coxackie virusenteroviridae vp1 pH 5 Gething et al., 1978 rhino virus vp1 pH 5internal fusion sequence enveloped viruses semliki forest v. togaviridaeE1 pH 5 White, 1990 sindbis virus White, 1990 unenveloped viruses rhesusrotavirus vp5 Mackow et al., 1988 Toxins of microorganism streptolysin oStreptococcus 69 kDa sulfhydryl activated, binds to cholesterol pH 7Kehoe et al., 1987 20-80 mer, 15 nm pores listeriolysin o L.monocytogenes 60 kDa sulfhydryl activated, binds to cholesterol pH 5Geoffroy et al., 1987 related to C9 and streptolysin o α-toxinStaphylococcus 34 kDa amphipathic surface, β-sheet structure, pH 7hexamer lesions, 2 nm pores hemolysin E. coli 416 AA 8 amphip. helixespH 7 Oropeza-Werkerle et al., 1992 hemolysin Trypanosoma cruzi 75 kDaanalogy to perforins, related to C9 pH 5 Andrews et al., 1990 Vertebrateimmunsystem perforin cytotoxic T-cells Ca²⁺ dependent membrane insertionOjcius and Jung, 1991 complement C9 (MAC c5b-8, 9₁₋₄) hollow proteincylinder, 10 nm channel, Shakdi und Tranum- Jensen, 1991; Easer, 1991highly regulated activity Sperm-egg fusion protein PH-30 α-subunit ofsurface protein, internal fusion sequence pH 7 Blobel et al., 1992Membrane-active Peptides Defense toxins melittin bea venom 26AA amph.α-helix Pro-kinked Blondelle und Houghten, 1991: Dempsey et al., 1991:Ikura und Inagaki, 1991 bombolitin bumblebee venom 17AA amph. α-helixArgiolas und Pisano, 1985 mastoparan wasp venom 14AA amph. α-helixArgiolas und Pisano, 1985 crabrolin hornet venom 13AA amph. α-helixArgiolas und Pisano, 1985 pardaxin moses sole fish 33AA amph. α-helixPro-kinked Shai et al., 1990 (shark repellant) Antibacterial peptidessarcotoxin IA flesh fly (in hemolymph) cecropins insects 37AA amph.α-helix Pro-kinked Easer, 1991 (humorel immune system, silk moth)maganin skin Xenopus laevis 23AA amph. α-helix Marion et al., 1998alameticin fungus 15-24AA amph. α-helix α-aminobutyric acid Easer, 1991(Trichoderma viride) Bacterial toxins d-toxin Staphylococcus aureus 26AAamph. α-helix acid induced Thiaudiere et al., 1991: Alouf et al., 1989amoebapore Entamoeba histolytica 25AA amph. α-helix acid induced Leippeet al., 1991 Vertebrate immune system defensins polynucleated 29-34AASS-bridged β-sheet Lehrer et al., 1991 neutrophils

TABLE 3 Transfection of BNL CL2 cells (6 μg pCMV-L DNA, 4 μg TfpL290)pLys 0 μg 0.3 μg 1 μg 3 μg 10 μg 20 μg 30 μg  0 μg P50 dim 160 330 540300 GLF 490 290 340 Melittin 0 0 0 70 425  4 μg P50 dim 3100 200 430 180410 GLF 670 600 170 EALA 3140 150 560 10 μg P50 dim 5700 760 13303424800 GLF 1950 16600 217000 215000 1980 EALA 2120 16800 179300 18170076360 20 μg P50 dim 3200 23300 185100 7054800 9344000 GLF 418400 320600294200 EALA 191000 181000 273600 Melittin 6545 Desoxy- 6730 34700 16000cholic acid Oleic acid 12200 11900 4100

13 23 amino acids amino acid single both peptide not provided 1 Gly LeuPhe Glu Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 MetIle Asp Gly Gly Gly Cys 20 23 amino acids amino acid single both peptidenot provided 2 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly TrpGlu Gly 1 5 10 15 Met Ile Asp Gly Gly Gly Cys 20 26 amino acids aminoacid single both peptide not provided 3 Met Ala Gln Asp Ile Ile Ser ThrIle Gly Asp Leu Val Lys Trp Ile 1 5 10 15 Ile Asp Thr Val Asn Lys PheThr Lys Lys 20 25 36 amino acids amino acid single both peptide notprovided 4 Met Ala Gln Asp Ile Ile Ser Thr Ile Gly Asp Leu Val Lys TrpIle 1 5 10 15 Ile Asp Thr Val Asn Lys Phe Thr Lys Lys Lys Lys Lys LysLys Lys 20 25 30 Lys Lys Lys Lys 35 34 amino acids amino acid singleboth peptide not provided 5 Trp Glu Ala Ala Leu Ala Glu Ala Leu Ala GluAla Leu Ala Glu His 1 5 10 15 Leu Ala Glu Ala Leu Ala Glu Ala Leu GluAla Leu Ala Ala Gly Gly 20 25 30 Ser Cys 35 amino acids amino acidsingle both peptide not provided 6 Gly Leu Phe Gly Ala Leu Ala Glu AlaLeu Ala Glu Ala Leu Ala Glu 1 5 10 15 His Leu Ala Glu Ala Leu Ala GluAla Leu Glu Ala Leu Ala Ala Gly 20 25 30 Gly Ser Cys 35 35 amino acidsamino acid single both peptide not provided 7 Gly Leu Phe Gly Ala LeuAla Glu Ala Leu Ala Glu Ala Leu Ala Glu 1 5 10 15 Ala Leu Ala Glu AlaLeu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly 20 25 30 Gly Ser Cys 35 34amino acids amino acid single both peptide not provided 8 Gly Leu PheGlu Leu Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala 1 5 10 15 Leu AlaGlu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly 20 25 30 Ser Cys34 amino acids amino acid single both peptide not provided 9 Gly Leu PheGly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 Leu AlaGlu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly 20 25 30 Ser Cys34 amino acids amino acid single both peptide not provided 10 Gly LeuPhe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15 LeuAla Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Ala Gly Gly 20 25 30 SerCys 23 amino acids amino acid single both peptide not provided 11 GlyLeu Phe Glu Ala Ile Glu Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15Met Ile Asp Gly Gly Gly Cys 20 36 amino acids amino acid single bothpeptide not provided 12 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr GlyLeu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln LysLys Lys Lys Lys Lys 20 25 30 Lys Lys Lys Lys 35 36 amino acids aminoacid single both peptide not provided 13 Gly Ile Gly Ala Val Leu Glu ValLeu Glu Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg LysArg Gln Gln Lys Lys Lys Lys Lys Lys 20 25 30 Lys Lys Lys Lys 35

What is claimed is:
 1. A transfection kit, comprising a carrier meanshaving in close confinement therein two or more container means, whereina first container means contains a substance having an affinity fornucleic acid, which substance is optionally coupled with aninternalizing factor for a higher eucaryotic cell; and a secondcontainer means contains an agent which has the ability per se of beinginternalized into a higher eucarvotic cell and of releasing the contentsof the cell's endosome into the cell's cytoplasm.
 2. A transfection kit,comprising a carrier means having in close confinement therein one ormore container means, wherein a first container means contains asubstance having an affinity for nucleic acid, which substance isoptionally coupled with an internalizing factor for a higher eucaryoticcell, and wherein a second container means contains a substance havingan affinity for a nucleic acid which is coupled to an agent which hasthe ability of being internalized into a higher eucalyotic cell as acomponent of a nucleic acid complex, and of releasing the contents ofthe cell's endosome, in which the complex is located after entering thecell, into the cell's cytoplasm.
 3. A transfection kit of claim 2,characterized in that it contains a first container means containing atransglutaminasecoupled adenovirus-polylysine conjugate.
 4. Atransfection kit, comprising a carrier means having in close confinementtherein one or more container means, wherein a first container meanscontains a biotin-modified endosomolytic agent and a second containermeans contains a streptavidin-modified substance having affinity fornucleic acid.
 5. A transfection kit of claim 4, characterized in thatsaid first container means contains biotinylated adenovirus and saidsecond container means contains streptavidin-polylysine.